Protein mutants that enhance the DNA cleavage activity of Acidaminococcus sp. CPF1

ABSTRACT

The present disclosure concerns polynucleotides and amino acids of  Acidaminococcus  sp. Cas12a (Cpf1) and methods for their use for genome editing in eukaryotic cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. 119 to U.S.Provisional Patent Application Ser. No. 62/870,268, filed Jul. 3, 2019and entitled “OPTIMIZED CAS12A (CPF1) PROTEINS FOR EFFICIENT GENOMEEDITING IN EUKARYOTIC CELLS,” U.S. Provisional Patent Application Ser.No. 62/749,607, filed Oct. 23, 2018 and entitled “DEEP-SCANNINGMUTAGENESIS UNCOVERS NOVEL MUTATIONS THAT ENHANCE THE DNA CLEAVAGEACTIVITY OF ACIDAMINOCOCCUS SP. CAS12A/CAS12A AT NON-CANONICAL TTTT PAMSITES” and U.S. Provisional Patent Application Ser. No. 62/716,138,filed Aug. 8, 2018 and entitled “NOVEL MUTATIONS THAT ENHANCE THE DNACLEAVAGE ACTIVITY OF ACIDAMINOCOCCUS SP. CPF1,” the contents of eachapplication are herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention pertains to the ability to cleave double-stranded DNA ofliving organisms at precise positions with the CRISPR/Cas12a (Cpf1)nuclease system. In particular, a series of recombinant Cas12a proteinsare described that are useful in a eukaryotic cell context.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Apr. 7, 2022, isnamed IDT01-013-PCT_Corrected_ST25.txt, and is 2,548,610 bytes in size.

BACKGROUND OF THE INVENTION

Cas12a is an RNA-guided endonuclease found in bacterial speciesincluding Acidaminococcus sp. and is part of the Clustered RegularlyInterspaced Short Palindromic Repeat (CRISPR) adaptive immune system.Cas12a is guided to a 21˜24-nt DNA target sequence, or commonly referredas protospacer, by a target site-specific 21˜24-nt complementary RNA.The Cas12a-gRNA ribonucleoprotein (RNP) complex mediates double-strandedDNA breaks (DSBs), which are then repaired by either the non-homologousend joining (NHEJ, typically introduces mutations or indels at the cutsite), or the homology directed repair (HDR) system for precise editingif a suitable template nucleic acid is present.

Critical to the recognition of correct DNA target for Cas12a includesboth crRNA and the canonical “TTTV” protospacer adjacent motif (PAM),which is a 4-bp sequence immediately upstream of the protospacer.Compared to the 2-bp NGG PAM of Cas9 from Streptococcus pyogenes, Cas12aexpanded the targetable loci in genome editing, particularly over theAT-rich sites that are inaccessible to the Cas9 system. However, due toits relatively low enzymatic activity, the likelihood that efficientgenome editing can be achieved for a given site is much lower than thatof the Cas9 system, which restricts its broader application. As theconsequence, the Cas12a system is frequently considered as analternative approach only when the genomic site is not targetable byCas9.

Protein engineering by mutagenesis can alter the preference of PAMsequence of CRISPR system. Through a structural-guided mutagenicscreening of residues in proximity of PAM sequence, previous study hasidentified two AsCpf1 variants that are compatible with TYCV and TATVPAMs, respectively. Although these variants collectively expanded thetargetable sites of Cpf1 system over the coding region of the humangenome by 3-fold, the utility of each individual variant is stilllimited, due to their mutually exclusive requirement of PAM sequences(TYCV vs TATV vs TTTV). Identifying Cpf1 variants with shorter PAM andgreater sequence flexibility without sacrificing the activity atcanonical PAM sites is highly desirable.

Thus, there is a need to enhance the utility of Cas12a. One aspect ofthe present application is to enhance the utility of Cpf1 by broadeningits PAM compatibility. In this regard, certain novel AsCas12a variantswith enhanced activity have been discovered. Another desired objectiveis to maximize delivery of the bacterial protein to the eukaryoticnucleus while simultaneously avoiding disruption of basic Cas12afunction. Since the Cas12a is a bacterial protein, certain moleculargenetic obstacles must first be overcome before one can achievesuccessful delivery of the protein to eukaryotic cells. This inventionprovides unique solutions to achieving these objectives.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, A CRISPR-associated protein comprising a polypeptideencoding a variant of AsCpf1 is provided. The variant of AsCpf1 isselected from the group consisting of M537R (SEQ ID NO.: 472), F870L(SEQ ID NO.: 473), and M537R/F870L (SEQ ID NO.: 465).

In a second aspect, a CRISPR ribonucleoprotein complex is provided. TheCRISPR ribonucleoprotein complex includes a guide RNA and aCRISPR-associated protein. The CRISPR-associated protein including apolypeptide encoding a variant of AsCpf1. The variant of AsCpf1 isselected from the group consisting of M537R (SEQ ID NO.: 472), F870L(SEQ ID NO.: 473), and M537R/F870L (SEQ ID NO.: 465).

In a third aspect, a method of increasing efficiency of gene editing atTTTN PAM sites in a cell with a CRISPR ribonucleoprotein complex isprovided. The method includes the step of contacting a cell with theCRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complexincludes a guide RNA and a CRISPR-associated protein. TheCRISPR-associated protein including a polypeptide encoding a variant ofAsCpf1. The variant of AsCpf1 is selected from the group consisting ofM537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473), and M537R/F870L (SEQID NO.: 465).

In a fourth aspect, a kit comprising a guide RNA and a CRISPR-associatedprotein is provided. The CRISPR-associated protein includes apolypeptide encoding a variant of AsCpf1.

In a fifth aspect, CRISPR-associated protein comprising a polypeptideencoding a variant of AsCas12a, wherein the variant of AsCas12a isselected from the group consisting of at least one variant amino acidselected from amino acid positions 499-640 and 840-913, provided thatthe variant AsCas12a provides an improvement inCRISPR/AsCas12a-associated nuclease activity at non-canonical TTTT PAMsites.

In a sixth aspect, a CRISPR ribonucleoprotein complex is provided. TheCRISPR ribonucleoprotein complex includes a guide RNA and aCRISPR-associated protein. The CRISPR-associated protein includes apolypeptide encoding a variant of AsCas12a, wherein the variant ofAsCas12a is selected from the group consisting of at least one variantamino acid selected from amino acid positions 499-640 and 840-913,provided that the variant AsCas12a provides an improvement inCRISPR/AsCas12a-associated nuclease activity at non-canonical TTTT PAMsites.

In a seventh aspect, a method of increasing efficiency of gene editingat non-canonical TTTT PAM sites in a cell with a CRISPRribonucleoprotein complex is provided. The method includes a step ofcontacting a cell with the CRISPR ribonucleoprotein complex thatincludes a guide RNA and a CRISPR-associated protein. TheCRISPR-associated protein includes a polypeptide encoding a variant ofAsCas12a, wherein the variant of AsCas12a is selected from the groupconsisting of at least one variant amino acid selected from amino acidpositions 499-640 and 840-913, provided that the variant AsCas12aprovides an improvement in CRISPR/AsCas12a-associated nuclease activityat non-canonical TTTT PAM sites.

In an eighth aspect, a kit including a guide RNA and a CRISPR-associatedprotein comprising a polypeptide encoding a variant of AsCas12a isprovided. The variant of AsCas12a is selected from the group consistingof at least one variant amino acid selected from amino acid positions499-640 and 840-913, provided that the variant AsCas12a provides animprovement in CRISPR/AsCas12a-associated nuclease activity atnon-canonical TTTT PAM sites.

In a ninth aspect, a nucleic acid encoding a CRISPR-associated proteincomprising a polypeptide encoding a variant of AsCas12a is provided. Thevariant of AsCas12a is selected from the group consisting of at leastone variant amino acid selected from amino acid positions 499-640 and840-913, provided that the variant AsCas12a provides an improvement inCRISPR/AsCas12a-associated nuclease activity at non-canonical TTTT PAMsites.

In a tenth aspect, a polynucleotide sequence encoding a Cas12apolypeptide is provided. The polynucleotide sequence includes one memberselected from the group consisting of SEQ ID NOs.: 5-17.

In an eleventh aspect, an amino acid sequence encoding a Cas12apolypeptide is provided. The amino acid sequence includes one memberselected from the group consisting of SEQ ID NOs.: 18-30.

In a twelfth aspect, a CAS endonuclease system comprising an expressioncassette encoding a polynucleotide sequence encoding a Cas12apolypeptide is provided. The includes one member selected from the groupconsisting of SEQ ID NOs.: 5-17.

In a thirteenth aspect, CAS endonuclease system comprising an amino acidsequence encoding a Cas12a polypeptide is provided The amino acidsequence includes one member selected from the group consisting of SEQID NOs.: 18-30.

In a fourteenth aspect, a method of performing genome editing in aeukaryotic cells is provided. The method includes the step ofintroducing an CAS endonuclease system into the eukaryotic cell, saidCAS endonuclease system comprising an expression cassette encoding apolynucleotide sequence encoding a Cas12a polypeptide, comprising onemember selected from the group consisting of SEQ ID NOs.: 5-17.

In a fifteenth aspect, a method of performing genome editing in aeukaryotic cell is provided. The method includes the step of introducingan CAS endonuclease system into the eukaryotic cell, said CASendonuclease system comprising an amino acid sequence encoding a Cas12apolypeptide comprising one member selected from the group consisting ofSEQ ID NOs.: 18-30.

In a sixteenth aspect, an CRISPR-associated protein comprising a fusionpolypeptide is provided. The fusion polypeptide includes an AsCas12aopen reading frame, a nuclear localization signal, optionally an aminoacid linker and optionally an affinity tag.

In a seventeenth aspect, a method of performing genome editing in aeukaryotic cell is provided. The method includes the step of introducingan CAS endonuclease system into the eukaryotic cell, said CASendonuclease system comprising a CRISPR-associated protein of accordingto the sixteenth aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts multiple recombinant forms of Cas12a yields a spectrum ofediting efficiencies. A series of recombinant Cas12a proteins withdiffering composition and arrangement of NLS sequences, purificationtags, and linkers (A-M; corresponding to SEQ ID NOs.:18-30) were clonedand purified to homogeneity. The resulting purified Cas12a derivativeswere delivered into HEK293 cells by electroporation complexed to RNAguides that target the HPRT-38186 (SEQ ID NO.:1) and HPRT-38228 loci(SEQ ID NO.:2). DNA was isolated from genome editing experiments 48 hrlater, and editing efficiencies were determined by PCR amplification ofedited loci using HPRT-FWD (SEQ ID NO.:3) and HPRT-REV (SEQ ID NO.:4)primers and the Alt-R Genome Editing Detection Kit (Integrated DNATechnologies). The following abbreviations apply for the illustratedconstructs: V5 refers to V5 epitope tag (SEQ ID NO.: 485); SV40 refersto a nuclear localization signal from simian virus SV40 large tumorantigen (SEQ ID NO.: 475); Cas12a refers to a Cas12a coding sequence;HIS refers to a hexahistidine tag (SEQ ID NO.: 487); OpT refers to anoptimized nuclear localization signal (SEQ ID NO.: 477); aNLS refers toan alternative nuclear localization signal(SEQ ID NO.: 479); BIP1 refersto a first bipartite nuclear localization signal (SEQ ID NO.: 481); andBIP2 refers to a second bipartite nuclear localization signal (SEQ IDNO.: 483). The arrows provided in the constructs depict transcriptionalstart sites for mRNA transcripts originating from the DNA.

FIG. 2 shows exemplary results of M537R and F870L mutations withenhanced the cleavage activity of Cpf1 in a bacterial-based activityassay. The screening E. coli strains were transformed with Cpf1expression vectors and the crRNA targeting HPRT-38346 site on the toxinexpression plasmid. The apparent activity of Cpf1 for TTTT or TTTC PAMcan be estimated by the number of survived colonies under arabinoseselection when equivalent amount of plasmids is delivered. Clearly, bothmutations increased the survival rate at TTTC and TTTT PAM sites,indicating an improved cleavage activity over the WT-Cpf1.

FIG. 3A shows an exemplary SDS-PAGE analysis of AsCpf1 variants used inthe in vitro cleavage assay and subsequent genome editing in human celllines. Indicated quantity of protein was loaded, and no difference wasobserved comparing to the WT-Cpf1. These results demonstrate M537R andF870L mutations can increase the in vitro cleavage activity of Cpf1 atnon-conical TTTT PAM site while maintaining high activity at canonicalTTTV site.

FIG. 3B shows exemplary DNA cleavage activities of Cpf1 variants atHPRT-38346 site with TTTT PAM or TTTC PAM(5′-TTTTACATAAAACTCTTTTAGGTTA-3′ (SEQ ID NO.: 494) and5′-TTTCACATAAAACTCTTTTAGGTTA-3′ (SEQ ID NO.: 495), respectively). Bothvariants resulted in a higher percentage of DNA cleavage at TTTT PAMsite than WT-Cpf1 (TTTC PAM). These results demonstrate M537R and F870Lmutations can increase the in vitro cleavage activity of Cpf1 atnon-conical TTTT PAM site while maintaining high activity at canonicalTTTC site.

FIG. 4 shows a summary of results showing exemplary results of M537R andF870L mutations having broadly enhanced the genomic targeting efficiencyof AsCpf1 at TTTN PAM sites in human cell line. The genomic targetingefficiencies of Cpf1 variants were tested in a human cell line modelusing T7 endonuclease I assay (T7EI). Twenty-four crRNAs targeting theCTNNB1 gene with TTTN PAMs were synthesized, assembled with Cpf1variants as RNP complex, and delivered by nucleofection (Lonza). Thegenomic DNA was collected 48 hours post-delivery to assess the formationof indels by T7EI. Not only enabled DNA cleavage at all TTTT PAM sitesthat many were not targetable by WT-Cpf1, the reported variants,particularly the double-mutant (M537R/F870L), broadly enhanced thetargeting efficiency of Cpf1 at 22 of 24 tested sites regardless of PAMsequence.

FIG. 5A shows exemplary correlation of relative survival rate ofM537R/F870L-AsCas12a under condition 1 (X-axis) vs. relative survivalrate of M537R/F870L-AsCas12a condition 2 (Y-axis). Consistent phenotypemeasurement was obtained (ρ˜0.7).

FIG. 5B shows exemplary correlation of relative survival rate ofM537R/F870L-AsCas12a under condition 1 (X-axis) vs. relative survivalrate of WT-AsCas12a under condition 3 (Y-axis). Consistent phenotypemeasurement was obtained (ρ˜0.7).

FIG. 5C shows exemplary correlation of relative survival rate ofM537R/F870L-AsCas12a under condition 2 (X-axis) vs. relative survivalrate of WT-AsCas12a under condition 2 (Y-axis). Consistent phenotypemeasurement was obtained (ρ˜0.7).

FIG. 5D shows exemplary variant scores of phenotype (relative survivalrate) of single point mutations of a selected number of AsCas12a atpositions 537 and 870 under conditions 1 and 2.

FIG. 5E shows exemplary variant scores of phenotype (relative survivalrate) of single point mutations of a selected number of AsCas12a atpositions 537 and 870 under conditions 1 and 2. FIGS. 5D and 5E showthat M537R and F870L are the optimal substitutions at respectivepositions, which is consistent with the result of our previous screeningand mutant characterization.

FIG. 5F shows exemplary variant scores of phenotype (relative survivalrate) of single point mutations of a selected number of AsCas12a atpositions 505, 510, 569 and 599 under conditions 1, 2 and 3.

FIG. 6A shows survival rate of E. coli resulting from DNA cleavageactivity of WT-AsCas12a at TTTT PAM. The cleavage activity of AsCas12avariants at TTTT PAM site was measured by bacterial-based activityassay. The petri dish plated with the initial bacterial plating is shownon the left, while the petri dish having the surviving bacterialcolonies is shown on the right. The survival of E. coli under selectionis dependent on the success cleavage of a toxin-expressing plasmidcontaining a TTTT PAM site.

FIG. 6B shows survival rate of E. coli resulting from DNA cleavageactivity of L505K-AsCas12a variant at TTTT PAM. The presentation ofpetri dishes and experimental details are as set forth in FIG. 6A.

FIG. 6C shows survival rate of E. coli resulting from DNA cleavageactivity of S510L-AsCas12a variant at TTTT PAM. The presentation ofpetri dishes and experimental details are as set forth in FIG. 6A.

FIG. 6D shows survival rate of E. coli resulting from DNA cleavageactivity of M537R-AsCas12a variant at TTTT PAM. The presentation ofpetri dishes and experimental details are as set forth in FIG. 6A.

FIG. 6E shows survival rate of E. coli resulting from DNA cleavageactivity of P569D-AsCas12a variant at TTTT PAM. The presentation ofpetri dishes and experimental details are as set forth in FIG. 6A.

FIG. 6F shows survival rate of E. coli resulting from DNA cleavageactivity of P599G-AsCas12a variant at TTTT PAM. The presentation ofpetri dishes and experimental details are as set forth in FIG. 6A.

FIG. 7A shows survival rate of E. coli resulting from DNA cleavageactivity of M537R/F870L-AsCas12a at TTTT PAM. The cleavage activity ofM537R/F870L-AsCas12a variants at TTTT PAM site was measured bybacterial-based activity assay. The petri dish plated with the initialbacterial plating is shown on the left, while the petri dish having thesurviving bacterial colonies is shown on the right. The survival of E.coli under selection is dependent on the success cleavage of atoxin-expressing plasmid containing a TTTT PAM site.

FIG. 7B shows survival rate of E. coli resulting from DNA cleavageactivity of L505K/M537R/F870L-AsCas12a variant at TTTT PAM. Thepresentation of petri dishes and experimental details are as set forthin FIG. 7A.

FIG. 7C shows survival rate of E. coli resulting from DNA cleavageactivity of M537R/F870L-AsCas12a variant at TTTT PAM. The presentationof petri dishes and experimental details are as set forth in FIG. 7A.

FIG. 7D shows survival rate of E. coli resulting from DNA cleavageactivity of P569D/M537R/F870L-AsCas12a variant at TTTT PAM. Thepresentation of petri dishes and experimental details are as set forthin FIG. 7A.

FIG. 7E shows survival rate of E. coli resulting from DNA cleavageactivity of P599G/M537R/F870L-AsCas12a variant at TTTT PAM. Thepresentation of petri dishes and experimental details are as set forthin FIG. 7A.

FIG. 7F shows survival rate of E. coli resulting from DNA cleavageactivity of S510L/M537R/F870L-AsCas12a variant at TTTT PAM. Thepresentation of petri dishes and experimental details are as set forthin FIG. 7A.

DETAILED DESCRIPTION

The present invention concerns compositions of Cas12a variants andmethods to enhance the utility of Cas12a and variants thereof forexpression in eukaryotic cells.

Definitions

When introducing elements of aspects of the disclosure or particularembodiments, the articles “a,” “an,” “the,” and “said” are intended tomean that there are one or more of the elements. The terms “comprising,”“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Theterm “or” means any one member of a particular list and also includesany combination of members of that list, unless otherwise specified.

As intended herein, the terms “substantially,” “approximately,” and“about” and similar terms are intended to have a broad meaning inharmony with the common and accepted usage in the art to which thesubject matter of this disclosure pertains. It should be understood bythose of skill in the art who review this disclosure that these termsare intended to allow a description of certain features described andclaimed without restricting the scope of these features to precisenumerical ranges provided. Accordingly, these terms should beinterpreted as indicating that insubstantial or inconsequentialmodifications or alterations of the subject matter described and claimedare considered to be within the scope of the invention as recited in theappended claims.

Definitions pertaining to certain terms and phrases applicable hereinmay be found in related US patents and publications, such as U.S. patentapplication Ser. Nos. 14/975,709, 15/299,549, 15/299,590, 15/299,593,15/881,684, 15/729,491, 15/821,736, 15/964,041, 15/839,817, 15/839,820,62/716,138, and U.S. Pat. No. 9,840,702.

The term “substantially purified,” as applied to a composition, refersto a composition having at least 90% purity or greater, including 90%purity, 95% purity, 99% purity and greater than 99% purity.

The adjective “isolated,” when modifying a composition, such as apolynucleotide, a polypeptide or a ribonucleoprotein complex refers to asubstantially purified composition, or in the case of aribonucleoprotein complex, at least one component being a substantiallypurified component. In further respect to an isolated ribonucleoproteincomplex, preferably all components are substantially purified.

The terms “nucleic acid” and “polynucleotide” are interchangeable havethe same meaning.

The terms “amino acid sequence,” “polypeptide,” and “protein” areinterchangeable have the same meaning.

The term “affinity tag” refers to a ligand that permits detection and/orselection of an oligonucleotide sequence to which the ligand isattached. For the purposes of this disclosure, a bait may include anaffinity tag. In particular, the affinity tag is positioned typically ateither or both the N′-terminus and/or C′-terminus of a polypeptidethrough the use of conventional chemical coupling technology orrecombinant DNA technology. Exemplary affinity tags include biotin,digoxigenin, streptavidin, polyhistine (for example, (His₆),),glutathione-S-transferase (GST), HaloTag®, AviTag, Calmodulin-tag,polyglutamate tag, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag 3,V5 tag, Xpress tag, a hapten, among others.

The term “eukaryotic cell,” includes those cells of or derived from aparticular organism, such as a plant or a mammal, including but notlimited to human, or non-human eukaryote or animal or mammal as hereindiscussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammalor primate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. Preferred humancells include cells derived from somatic cells and germ line cells.Exemplary somatic cells include cells from every major organ and tissuesystem, including the immune system and hematopoietic system.

As set forth herein, the Conditions 1, 2 and 3 refer to differentcombinations of background strain and the amounts of gRNA introduced inthe background strain before selection of the variants. Condition 1 is aM537R/F870L background that includes an introduced amount of gRNA (100pmol per 10 microliter transformation/plating experiment) in whichvariants were selected. Condition 2 is a M537R/F870L background thatincludes an introduced amount of gRNA (50 pmol per 10 microlitertransformation/plating experiment) in which variants were selected.Condition 3 is a Wild-type AsCpf1 background that includes an introducedamount of gRNA (200 pmol per 10 microliter transformation/platingexperiment) in which variants were selected.

Cas12a Polypeptides Having Eukaryotic Nuclear Localization Signals

Since Cas12a is a bacterial protein, it has no native targetingmechanism to reach the eukaryotic nucleus, where the target DNA resides.

To more efficiently target proteins to the eukaryotic nucleus, shortprotein sequences called nuclear localization signals (NLS) are commonlyadded to the amino- or carboxy-terminal ends of a given open readingframe. NLSs are recognized by import proteins on the eukaryotic nuclearenvelope that first bind to the nuclear membrane, and subsequentlypermit pore translocation into the nucleus by an energy-dependentprocess. While recombinant protein tags like an NLS can greatly improvelocalization, any addition of exogenous amino acid sequences stands areasonable chance of perturbing protein function. As such, discovering arecombinant Cas12a protein sequence that facilitates the highest amountof nuclear delivery without negatively affecting its activity willultimately result in the most potent Cas12a genome editing solution,which is non-trivial and highly desirable.

To improve nuclear delivery of Cas12a without perturbing its function,several different recombinant versions of Cas12a were constructed inwhich the identity, location, and number of protein tags (NLS,hexahistidine tag (an exemplary affinity tag)) were varied. Whereashexahistidine and V5 tags were added to Cas12a constructs to aid inprotein purification and/or detection, the NLS tags were added to assistin delivery to the eukaryotic nucleus. Domain-breaking linker sequenceswere also varied in composition and location to empirically determinethe best arrangement and context of tagged sequences. All constructswere first expressed in E. coli, and recombinant Cas12a proteins werepurified with immobilized metal affinity chromatography (IMAC) followedby ion exchange chromatography as described previously.

AsCpf1 Polypeptide Variants Having Novel Cleavage Activities

Amino acid substitutions in AsCpf1 that enhance the cleavage activity atboth canonical (TTTV) and non-canonical (TTTT) PAM sites using abacterial screening approach are described. This screen contained twocomponents: i) a toxin plasmid encoding an arabinose-inducible cellproliferation toxin and a CRISPR/Cpf1 on-target cleavage site(HPRT-38346) with TTTT PAM, and ii) a chloramphenicol resistance plasmidcontaining a randomly-mutagenized region within the AsCpf1 sequence (˜5mutations per kb). The screen was performed as follows: E. coliBW25141(λDE3) was transformed with the toxin plasmid containing theHPRT-38346 target site in the absence of arabinose, where the toxin isnot produced and cell survival is permitted. Cells with stablyreplicating toxin plasmid are then transformed with the AsCpf1expression plasmid and crRNA targeting HPRT-38346, and then cells wereplated on media containing both chloramphenicol and arabinose. Bacteriathat grew were those that i) successfully transformed with the AsCpf1expression plasmid, ii) expressed sufficient AsCpf1 variant to cleavethe toxin plasmid at HPRT-38346 site using TTTT PAM. The AsCpf1expression plasmids within the survived cells were recovered and used inthe subsequent round of selection. After multiple rounds of selection,the identities of enriched AsCpf1 variants were determined by DNAsequencing, and carried forward for analysis in mammalian cells.

The disclosure provides following two novel point mutations and thecombination in the AsCpf1 gene that enhances the cleavage activity:M537R and F870L. The cleavage activity of individual mutant was firsttested in a bacterial-based activity assay. Next, purified proteins werefurther tested in vitro and in human cell lines. In summary, bothsubstitutions significantly enhanced the DNA cleavage activity of Cpf1at TTTT PAM sites in all assays. Further, the combination of M537R andF870L broadly enhanced the targeting efficiency of AsCpf1 in human cellline. Overall, the present invention identifies novel amino-acidpositions in the AsCpf1 gene that can be mutated to improve its cleavageactivity at all TTTN (N=A/G/C/T) PAM sites.

As explained in the Background section, the prior art consists of usingwild-type Cpf1 protein or two variants that are compatible with TYCV andTATV PAMs. As stated previously, these variants have limited utility dueto the complex and mutually exclusive requirement of PAM sequence.Further, none of the variants showed any improved cleavage activity atTTTT PAM, which is unfortunately more frequent than other TTTV PAM sites(V=G/A/C) throughout the human genome. In contrast, not only enablingefficient cleavage at TTTT PAM, the mutations reported in this invention(M537R and F870L) broadly enhanced the cleavage activity of Cpf1 atcanonical TTTV sites tested in human cell line. Together, the enhancedactivity and broadened PAM flexibility (TTTN) of this invention makes ita superior CRISPR enzyme, which could directly replace the currentwild-type Cpf1 in the application genomic editing.

High-Throughput Generation of AsCas12a Variants Having Cleavage ActivityTowards a TTTT PAM-Containing Target Site.

The phenotype of all point mutations in the following regions ofAsCas12a: 499-640 and 840-913 in the bacterial screening measuring theDNA cleavage activity at non-canonical TTTT PAM is described. Three setsof screening were performed to measure the phenotype of each pointmutation, in the background of both WT-AsCas12a andM537R/F870L-AsCas12a. Cross-comparison of three datasets revealedconsistent phenotype measurements, which enabled us to isolate novelAsCas12a variants with enhanced activity beyond M537R and F870L.

The high-throughput characterization of Cas12a activity at a TTTT PAMsite provided the functional consequence of every possible single aminoacid change within targeted region. The unbiased strategy of the presentinvention enables one to identify a large collection of mutants tofurther enhance the cleavage activity of AsCas12a over our previousinvention (M537R/F870L).

To improve the coverage and efficiency of the screening, we generated anAsCas12a deep-scanning mutagenesis library containing all possible pointmutations on the protein level in the targeted regions (490-640 and840-913), with most clones contain only one mutation. This type oflibrary allowed us to directly evaluate the phenotype of each pointmutation, by measuring their relative survival rates over the referenceprotein in the bacterial screen. Briefly, the screening strain harboringthe toxin plasmid was transformed with AsCas12a library with TTTTPAM-containing target site on the toxin plasmid. After transformation,cells were plated on selective media. AsCas12a expression plasmidscarried by the survived E. coli cells were extracted and purified. Bothinput and selected plasmid libraries were PCR amplified, and sequencedon Illumina MiSeq with 1˜2 million reads per library. The frequencies ofeach AsCas12a variant in both libraries were determined using Enrich 2,and normalized to the reference protein (WT or M537R/F870L). Therelative survival rate of each point mutation over reference wascalculated as the ratio of normalized frequency between selected andinput library. Since the degree of cell survival is indicative of theDNA cleavage activity of each AsCas12a variant, any variants with highersurvival rate than the reference protein would be those with enhancedactivity at TTTT PAM.

As presented herein, codon-optimized Cas12a polypeptides are provided,including codon-optimized Cas12a polypeptides for CRISPRribonucleoprotein complexes. An example of a codon-optimized sequence,is in this instance a sequence optimized for expression in eukaryotes,e.g., humans (i.e., being optimized for expression in humans), or foranother eukaryote, animal or mammal as herein discussed. Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a CRISPR Cas12a polypeptide is a codonoptimized for expression in particular cells, such as eukaryotic cells.The eukaryotic cells may be those of or derived from a particularorganism, such as a plant or a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded.

Preferred human cells include cells derived from somatic cells and germline cells. Exemplary somatic cells include cells from every major organand tissue system, including the immune system and hematopoietic system.

In general, codon optimization refers to a process of modifying anucleic acid sequence for enhanced expression in the host cells ofinterest by replacing at least one codon (e.g. about or more than about1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the nativesequence with codons that are more frequently or most frequently used inthe genes of that host cell while maintaining the native amino acidsequence. Various species exhibit particular bias for certain codons ofa particular amino acid. Codon bias (differences in codon usage betweenorganisms) often correlates with the efficiency of translation ofmessenger RNA (mRNA), which is in turn believed to be dependent on,among other things, the properties of the codons being translated andthe availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization. Codon usage tables are readily available. SeeNakamura, Y., et al. “Codon usage tabulated from the international DNAsequence databases: status for the year 2000” Nucl. Acids Res. 28:292(2000). Computer algorithms for codon optimizing a particular sequencefor expression in a particular host cell are also available, such asGene Forge (Aptagen; Jacobus, Pa.), are also available. In someembodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50,or more, or all codons) in a sequence encoding a Cas12a correspond tothe most frequently used codon for a particular amino acid.

Additionally, codon-optimized Cas12a polypeptides are provided,including codon-optimized Cas12a polypeptides for CRISPRribonucleoprotein complexes, wherein the Cas12a polypeptide sequenceoptimized for expression in prokaryotes, such as bacteria (e.g., E.coli).

Applications

This invention is useful for either basic research or therapeutic fieldsfor any CRISPR/Cas12a DNA cleavage and/or gene editing experiments ortreatments. The superior activity of these recombinant variants could beapplied to Cas12a from any species or potentially any CRISPR enzyme.

In a first aspect, A CRISPR-associated protein comprising a polypeptideencoding a variant of AsCpf1 is provided. The variant of AsCpf1 isselected from the group consisting of M537R (SEQ ID NO.: 472), F870L(SEQ ID NO.: 473), and M537R/F870L (SEQ ID NO.: 465). In a firstrespect, the CRISPR-associated protein corresponds to a variant ofAsCpf1 is M537R. In a second respect, the CRISPR-associated proteincorresponds to a variant of AsCpf1 is F870L (SEQ ID NO.: 473). In athird respect, the CRISPR-associated protein corresponds to a variant ofAsCpf1 is M537R/F870L (SEQ ID NO.: 465).

In a second aspect, a CRISPR ribonucleoprotein complex is provided. TheCRISPR ribonucleoprotein complex includes a guide RNA and aCRISPR-associated protein. The CRISPR-associated protein including apolypeptide encoding a variant of AsCpf1. The variant of AsCpf1 isselected from the group consisting of M537R (SEQ ID NO.: 472), F870L(SEQ ID NO.: 473), and M537R/F870L (SEQ ID NO.: 465). In a firstrespect, the CRISPR ribonucleoprotein complex includes the variant ofAsCpf1 being M537R. In a second respect, the CRISPR ribonucleoproteincomplex includes the variant of AsCpf1 being F870L (SEQ ID NO.: 473). Ina third respect, the CRISPR ribonucleoprotein complex includes thevariant of AsCpf1 being M537R/F870L (SEQ ID NO.: 465).

In a third aspect, a method of increasing efficiency of gene editing atTTTN PAM sites in a cell with a CRISPR ribonucleoprotein complex isprovided. The method includes the step of contacting a cell with theCRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complexincludes a guide RNA and a CRISPR-associated protein. TheCRISPR-associated protein including a polypeptide encoding a variant ofAsCpf1. The variant of AsCpf1 is selected from the group consisting ofM537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R/F870L (SEQ IDNO.: 465). In a first respect, the TTTN PAM sites consists of oneselected form the group of TTTA, TTTT and TTTC PAM sites.

In a fourth aspect, a kit includes a guide RNA and a CRISPR-associatedprotein is provided. The CRISPR-associated protein includes apolypeptide encoding a variant of AsCpf1. The variant of AsCpf1 isselected from the group consisting of M537R (SEQ ID NO.: 472), F870L(SEQ ID NO.: 473), and M537R/F870L (SEQ ID NO.: 465). In a firstrespect, the variant of AsCpf1 is M537R. In a second respect, thevariant of AsCpf1 is F870L (SEQ ID NO.: 473). In a third respect, thevariant of AsCpf1 is M537R/F870L (SEQ ID NO.: 465).

In a fifth aspect, a CRISPR-associated protein comprising a polypeptideencoding a variant of AsCas12a, wherein the variant of AsCas12a isselected from the group consisting of at least one variant amino acidselected from amino acid positions 499-640 and 840-913, provided thatthe variant AsCas12a provides an improvement inCRISPR/AsCas12a-associated nuclease activity at non-canonical TTTT PAMsites. In a first respect, the variant of AsCas12a is selected from thegroup consisting of SEQ ID NOs.: 59-245. In a second respect, thevariant of AsCas12a, as described in the first aspect or the foregoingfirst respect of the first aspect, further comprises mutations ofM537R/F870L (SEQ ID NO.: 465).

In a sixth aspect, a CRISPR ribonucleoprotein complex is provided. TheCRISPR ribonucleoprotein complex includes a guide RNA and aCRISPR-associated protein. The CRISPR-associated protein includes apolypeptide encoding a variant of AsCas12a, wherein the variant ofAsCas12a is selected from the group consisting of at least one variantamino acid selected from amino acid positions 499-640 and 840-913,provided that the variant AsCas12a provides an improvement inCRISPR/AsCas12a-associated nuclease activity at non-canonical TTTT PAMsites. In a first respect, the variant of AsCas12a is selected from thegroup consisting of SEQ ID NOs.: 59-245. In a second respect, thevariant of AsCas12a, as described in the second aspect or the foregoingfirst respect of the second aspect, further comprises mutations ofM537R/F870L (SEQ ID NO.: 465).

In a seventh aspect, a method of increasing efficiency of gene editingat non-canonical TTTT PAM sites in a cell with a CRISPRribonucleoprotein complex is provided. The method includes a step ofcontacting a cell with the CRISPR ribonucleoprotein complex thatincludes a guide RNA and a CRISPR-associated protein. TheCRISPR-associated protein includes a polypeptide encoding a variant ofAsCas12a, wherein the variant of AsCas12a is selected from the groupconsisting of at least one variant amino acid selected from amino acidpositions 499-640 and 840-913, provided that the variant AsCas12aprovides an improvement in CRISPR/AsCas12a-associated nuclease activityat non-canonical TTTT PAM sites. In a first respect, the variant ofAsCas12a is selected from the group consisting of SEQ ID NOs.: 59-245.In a second respect, the variant of AsCas12a, as described in the thirdaspect or the foregoing first respect of the third aspect, furthercomprises mutations of M537R/F870L (SEQ ID NO.: 465).

In an eighth aspect, a kit including a guide RNA and a CRISPR-associatedprotein comprising a polypeptide encoding a variant of AsCas12a isprovided. The variant of AsCas12a is selected from the group consistingof at least one variant amino acid selected from amino acid positions499-640 and 840-913, provided that the variant AsCas12a provides animprovement in CRISPR/AsCas12a-associated nuclease activity atnon-canonical TTTT PAM sites. In a first respect, the variant ofAsCas12a is selected from the group consisting of SEQ ID NOs.: 59-245.In a second respect, the variant of AsCas12a, as described in the fourthaspect or the foregoing first respect of the fourth aspect, furthercomprises mutations of M537R/F870L (SEQ ID NO.: 465).

In a ninth aspect, a nucleic acid encoding a CRISPR-associated proteincomprising a polypeptide encoding a variant of AsCas12a is provided. Thevariant of AsCas12a is selected from the group consisting of at leastone variant amino acid selected from amino acid positions 499-640 and840-913, provided that the variant AsCas12a provides an improvement inCRISPR/AsCas12a-associated nuclease activity at non-canonical TTTT PAMsites. Highly preferred nucleic acids encoding a CRISPR-associatedprotein include isolated nucleic acids encoding a CRISPR-associatedprotein. In a first respect, the variant of AsCas12a is selected fromthe group consisting of SEQ ID NOs.: 59-245. In a second respect, thevariant of AsCas12a further includes mutations of M537R/F870L (SEQ IDNO.: 465). In a third respect, the nucleic acid is operably linked tosuitable transcription elements to express the nucleic acid. In a fourthrespect, the nucleic acid is DNA or RNA.

In a tenth aspect, a polynucleotide sequence encoding a Cas12apolypeptide is provided. The polynucleotide sequence includes one memberselected from the group consisting of SEQ ID NOs.: 5-17.

In an eleventh aspect, an amino acid sequence encoding a Cas12apolypeptide is provided. The amino acid sequence includes one memberselected from the group consisting of SEQ ID NOs.: 18-30.

In a twelfth aspect, a CAS endonuclease system comprising an expressioncassette encoding a polynucleotide sequence encoding a Cas12apolypeptide is provided. The includes one member selected from the groupconsisting of SEQ ID NOs.: 5-17.

In a thirteenth aspect, CAS endonuclease system comprising an amino acidsequence encoding a Cas12a polypeptide is provided The amino acidsequence includes one member selected from the group consisting of SEQID NOs.: 18-30.

In a fourteenth aspect, a method of performing genome editing in aeukaryotic cells is provided. The method includes the step ofintroducing an CAS endonuclease system into the eukaryotic cell, saidCAS endonuclease system comprising an expression cassette encoding apolynucleotide sequence encoding a Cas12a polypeptide, comprising onemember selected from the group consisting of SEQ ID NOs.: 5-17.

In a fifteenth aspect, a method of performing genome editing in aeukaryotic cell is provided. The method includes the step of introducingan CAS endonuclease system into the eukaryotic cell, said CASendonuclease system comprising an amino acid sequence encoding a Cas12apolypeptide comprising one member selected from the group consisting ofSEQ ID NOs.: 18-30.

In a sixteenth aspect, an CRISPR-associated protein comprising a fusionpolypeptide is provided. The fusion polypeptide includes an AsCas12aopen reading frame, a nuclear localization signal, optionally an aminoacid linker and optionally an affinity tag. Highly preferredCRISPR-associated proteins include isolated CRISPR-associated proteins.In a first respect, the AsCas12a open reading frame is selected from thegroup consisting of SEQ ID NOs.: 59-245. In a second respect, thenuclear localization signal is selected from SEQ ID NOs.: 475, 477, 479,481 and 483. In a third respect, the CRISPR-associated protein isencoded by SEQ ID NOs.: 488-491. In a fourth respect, theCRISPR-associated protein is selected from SEQ ID NOs.: 492 and 493.

In a seventeenth aspect, a method of performing genome editing in aeukaryotic cell is provided. The method includes the step of introducingan CAS endonuclease system into the eukaryotic cell, said CASendonuclease system comprising an CRISPR-associated protein of accordingto the sixteenth aspect. Highly preferred CRISPR-associated proteinsinclude isolated CRISPR-associated proteins.

EXAMPLES Example 1 Recombinant Cas12a Proteins with Varying Tags/LinkerSequences Yield a Spectrum of Editing Efficiencies

The following Example demonstrates that recombinant Cas12a proteins withonly modest changes in tag sequences at the amino- and carboxy-terminiresults in proteins that demonstrate a wide range of editingefficiencies in human cells (FIG. 1).

Briefly, the method of site directed mutagenesis (SDM) was employed tocreate the expression constructs having As Cas12a coding sequences withdifferent nuclear localization signals (NLS's). Site directedmutagenesis was performed by designing complimentary primers thatencompass the desired nucleotide base change(s), along with flankingplasmid vector sequence, wherein each flanking region has a meltingtemperature (T_(m)) of at least 60° C. A polymerase chain reaction (PCR)run was then performed using standard cycling conditions for a total of16 cycles. The restriction enzyme, DPN I, was added to digest away thestarting plasmid vector material so only the new product containing thebase changes remain. After DPN I treatment, a small amount of the PCRproduct was transformed into competent E. coli cells, recovered in SOCmedia and plated onto kanamycin resistance Luria Broth (LB) agar plates.Colonies were screened using the Sanger sequencing method to verifycorrect base changes in selected clones.

The results indicate that the ideal sequence and placement of NLSsequences on Cas12a is not obvious, and that a highly efficient Cas12agenome editing solution must be empirically determined as was done inthis study. Proteins were tested using guides that target the HPRT-38186(SEQ ID NO.:1) and HPRT-38228 (SEQ ID NO.:2) loci in human cells. SEQ IDNOs.: 1-4 are depicted in Table 1.

TABLE 1 Sequence of oligonucleotides used as crRNAs or PCR primers. NameSequence SEQ ID NO. HPRT- rUrArArUrUrUrCrUrArCrUrCrUrUr SEQ ID NO. 138186-S GrUrArGrArUrUrArArUrGrCrCr CrUrGrUrArGrUrCrUrCrUrCrUrG HPRT-rUrArArUrUrUrCrUrArCrUrCrUr SEQ ID NO. 2 38228-SUrGrUrArGrArUrUrArArUrUrArAr CrArGrCrUrUrGrCrUrGrGrUrGrA HPRT-FWDAAGAATGTTGTGATAAAAGGTGATGCT SEQ ID NO. 3 HPRT-REVGAGGCAGAAGTCCCATGGATGTGT SEQ ID NO. 4

The following nucleotide sequences that encode preferred Cas12apolypeptides of this Example are depicted below:

SEQ ID NO.: 5ATGGGCAGCAGCAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCAGGTAAACCGATTCCGAATCCGCTGCTGGGTCTGGATAGCACCGCACCGAAAAAAAAACGTAAAGTTGGTATTCATGGTGTTCCGGCAGCAACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACCCTAAAAAAAAACGCAAAGTGAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 6ATGGGCAGCAGCAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCAGCACCGAAAAAAAAACGTAAAGTTGGTATTCATGGTGTTCCGGCAGCAACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACCCTAAAAAAAAACGCAAAGTGAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 7ATGCCGAAAAAAAAACGCAAAGTGGGTATTCATGGTGTTCCGGCAGCAACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACCCGAAAAAAAAACGCAAAGTGCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 8ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACCCGAAAAAAAAACGCAAAGTGCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 9ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCAACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTTGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 10ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTTGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 11ATGGGCAGCAGCAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCCAACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTTGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 12ATGCCGCCTCCGAAACGTCCGCGTCTGGATGGTATCCACGGAGTCCCAGCAGCCACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTTGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGASEQ ID NO.: 13ATGAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTTGGTATCCACGGAGTCCCAGCAGCCACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTTGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 14ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAAACGTCCGGCAGCAACCAAAAAAGCAGGTCAGGCAAAAAAGAAAAAAGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 15ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAAACGTACCGCAGATGGTAGCGAATTTGAAAGCCCGAAAAAAAAGCGTAAGGTGGAAGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 16ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTTGGTGGTAGCGGCGGTAGTAAACGTACCGCAGATGGTAGCGAATTTGAAAGCCCGAAAAAAAAGCGTAAGGTGGAAGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO.: 17ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGTAAACGTCCGGCAGCAACCAAAAAAGCAGGTCAGGCAAAAAAGAAAAAAGGTGGTAGCGGCGGTAGTAAACGTACCGCAGATGGTAGCGAATTTGAAAGCCCGAAAAAAAAGCGTAAGGTGGAAGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA

The following amino acid sequences of preferred Cas12a polypeptides aredepicted below.

SEQ ID NO.: 18 MGSSSSGLVPRGSHMASMTGGQQMGRDPGKPIPNPLLGLDSTAPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNPKKKRKVKLAAALEHHHHHH SEQ ID NO.: 19MGSSSSGLVPRGSHMASMTGGQQMGRDPAPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN PKKKRKVKLAAALEHHHHHHSEQ ID NO.: 20 MPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNPKKKRKVLEHHHHHH SEQ ID NO.: 21MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNPKKKRKVLEHHHHHH SEQ ID NO.: 22MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRDPTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH SEQ ID NO.: 23MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSG GSGGSLEHHHHHH SEQ IDNO.: 24 MGSSSSGLVPRGSHMASMTGGQQMGRDPTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH SEQ ID NO.: 25MPPPKRPRLDGIHGVPAATQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH SEQ ID NO.: 26MSSDDEATADSQHAAPPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH SEQ ID NO.: 27MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRKRPAATKKAGQAKKKKGGSGGSGGSGGSGGSGGSGGSGGSL EHHHHHH SEQ ID NO.:28 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRKRTADGSEFESPKKKRKVEGGSGGSGGSGGSGGSGGSGGSG GSLEHHHHHH SEQ IDNO.: 29 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSKRTADGSEFESPKKKRKVEGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH SEQ ID NO.: 30MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRKRPAATKKAGQAKKKKGGSGGSKRTADGSEFESPKKKRKVEGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH

Example 2 Novel Cpf1 Mutants Enhance the DNA Cleavage Activity at TTTTPAM Site in the Bacterial-Based Activity Assay

The following Example demonstrates the enhanced activity of theinvention at both TTTT and TTTC PAM sites in a bacterial-based activityassay (FIG. 2). The screening strains harboring the toxin plasmid weretransformed with WT, M537R or F870L AsCpf1 expression plasmid. Afterrecovery and IPTG induction, cells were plated on LB-Chloramphenicolmedia with or without arabinose. The degree of cell survival under thearabinose selection compared to the transformation input control(without arabinose) is indicative of the cleavage activity of Cpf1variants at the HPRT-38346 protospacer on the toxin plasmid, in thecontext of TTTT or TTTC PAM.

For WT-Cpf1, the survival rate of transformed E. coli at TTTC issignificantly higher than the TTTT PAM, which is in good agreement withthe prior knowledge that the TTTT is a low activity PAM site^([6]). Incontrast, both M537R and F870L increased the survival rate at TTTT PAM,indicating these mutants broadened the PAM compatibility of AsCpf1 atthis alternative PAM site. More importantly, the survival rate of bothmutants at the canonical TTTC PAM is even higher than the WT-Cpf1,suggesting these mutants generally enhanced the performance of AsCpf1protein at other TTTV sites as well. Given these positive results,individual AsCpf1 variant and the double mutant (M537R/F870L) wereexpressed and purified to determine their intrinsic cleavage activitiesin vitro.

Example 3 Novel Cpf1 Mutants Enhances the Intrinsic DNA CleavageActivity at TTTT PAM Sites In Vitro

The intrinsic DNA cleavage activities of AsCpf1 variants (M537R, F870Land M537R/F870L) were compared to the wild type protein using in vitrocleavage assay. Briefly, the Cpf1-crRNA ribonucleoprotein (RNP) complexwas first assembled by incubating the purified proteins (FIG. 3A) withHPRT-38346 crRNA in 1× cleavage buffer (20 mM HEPES, pH 7.5, 150 mM KCl,5 mM MgCl₂, 10% Glycerol, and 1 mM DTT) for 15 minutes at 37° C. Thecleavage reactions were initiated by titrating RNP complex (8˜500 nM) in10 nM dsDNA substrate containing the HPRT-38346 protospacer, in thecontext of TTTC or TTTT PAM. Cleavage reactions at various time pointswere sampled and quenched by 50 mM EDTA. After removing the AsCpf1protein by Proteanise K treatment (56° C., 30 minutes), reactions wereresolved using capillary electrophoresis (Fragment Analyzer, AATI). Therelative concentration of cleavage products and uncleaved dsDNA werequantified to calculate the percentage of DNA cleavage.

The intrinsic DNA cleavage activities of WT and Cpf1 variants at theTTTT and TTTC PAM sites (5′-TTTTACATAAAACTCTTTTAGGTTA-3′ (SEQ ID NO.:494) and 5′-TTTCACATAAAACTCTTTTAGGTTA-3′ (SEQ ID NO.: 495),respectively) were compared in FIG. 3B. Only a single RNP concentration(31 nM) at 20 seconds time-point was shown for simplicity. As expected,the single-nucleotide change of PAM sequence from TTTC to TTTT reducedthe cleavage activity of WT-Cpf1 from ˜95% to ˜40%. Consistent with theobservations in the bacterial-based activity assay, both mutantssignificantly increased the DNA cleavage at TTTT PAM, while maintaininghigh activity at TTTC PAM (FIG. 3B). The double-mutant (M537R/F870L) hassimilar activities to the M537R in this assay. However, it is worth tonote that this is likely due to the limited resolution of thisparticular assay, to resolve further differences among thosehigh-activity variants. Overall, these results demonstrated that thereported mutations improved the activity of Cpf1 by enhancing theintrinsic DNA cleavage. Therefore, we anticipate that the observedbenefits of these mutants will be broadly applicable and independent ofthe delivery methods and/or cellular contexts of the specificexperiment.

Example 4 Novel Mutants Broadly Enhance the Targeting Efficiency of TTTNPAM Sites in Human Cell Line

The following Example demonstrates the ability of the invention toincrease the efficiency of gene editing at TTTN PAM sites when theCpf1-crRNA complex is delivered into cells as an RNP.

CRISPR/Cpf1 cellular editing experiments were performed by first forming4 mM RNP complex with purified Cpf1 protein and the Alt-R™ crRNAs inOpti-MEM for 5 min at 25° C. The targeted protospacers and PAM sequencesin CTNNB1 loci are shown in Table 2. RNP complexes were then transfectedinto HEK293 cells by Lonza nucleofection. Experiments were performed inbiological triplicate. After 48 hr at 37° C. with 5% CO₂, adherent cellswere washed with 0.1 ml PBS and lysed with 0.05 ml QuickExtract' DNAextraction solution. Cell lysates were incubated at 65° C. for 15 minfollowed by heat-inactivation at 98° C. for 3 min. Crude DNA sampleswere then diluted 3-fold with 0.1 ml ddH₂O and used as PCR templates.PCR primers are indicated in Table 2. PCR was used to amplify 1 kbfragments of the CTNNB1 loci using the KAPA HiFi DNA Polymerase and thefollowing cycling parameters: 95^(5:00) (98^(0:20), 64^(0:15),72^(0:30)) repeated 29 times, 72^(2:00).

Heteroduplexes were formed using the following cycling parameters:95^(10:00) cooled to 85 over 1 min, 85^(1:00) cooled to 75 over 1 min,75^(1:00) cooled to 65 over 1 min, 65^(1:00) cooled to 55 over 1 min,55^(1:00) cooled to 45 over 1 min, 45^(1:00) cooled to 35 over 1 min,35^(1:00) cooled to 25 over 1 min, 25^(1:00). Heteroduplexes werecleaved by the addition of 2U T7 Endonuclease I (New England Biolabs)for 1 hr at 37 C, and cut products were analyzed by capillaryelectrophoresis (Fragment Analyzer, Advanced Analytical).

TABLE 2 Sequence of DNA target sites and primer used for PCRamplification (SEQ ID NOs.: 31-58). Name Sequence (5′-3′) SEQ ID NO.:HPRT38346 TTTTACATAAAACTCTTTTAGGTTA SEQ ID NO.: TTTT PAM 31 HPRT38346TTTCACATAAAACTCTTTTAGGTTA SEQ ID NO.: TTTC PAM 32 CTNNB1TTTTCCCCTCCCTGGCTTTTATTAT SEQ ID NO.: 111-S 33 CTNNB1TTTCCCCTCCCTGGCTTTTATTATT SEQ ID NO.: 112-S 34 CTNNB1TTTTATTATTACAACTCTGTGCTTT SEQ ID NO.: 127-S 35 CTNNB1TTTATTATTACAACTCTGTGCTTTT SEQ ID NO.: 128-S 36 CTNNB1TTTTTCATCACCATCCTGAATATCT SEQ ID NO.: 149-S 37 CTNNB1TTTTCATCACCATCCTGAATATCTA SEQ ID NO.: 150-S 38 CTNNB1TTTCATCACCATCCTGAATATCTAT SEQ ID NO.: 151-S 39 CTNNB1TTTATACTATTAATAAAAAGACATT SEQ ID NO.: 184-S 40 CTNNB1TTTATTAATAGTATAAATATTAATT SEQ ID NO.: 193-AS 41 CTNNB1TTTTATTAATAGTATAAATATTAAT SEQ ID NO.: 194-AS 42 CTNNB1TTTTTATTAATAGTATAAATATTAA SEQ ID NO.: 195-AS 43 CTNNB1TTTTTGGTAAGGAGGAGTTTTCACT SEQ ID NO.: 207-S 44 CTNNB1TTTTGGTAAGGAGGAGTTTTCACTG SEQ ID NO.: 208-S 45 CTNNB1TTTGGTAAGGAGGAGTTTTCACTGA SEQ ID NO.: 209-S 46 CTNNB1TTTTCACTGAAGTTCAGCAGTGATG SEQ ID NO.: 224-S 47 CTNNB1TTTCACTGAAGTTCAGCAGTGATGG SEQ ID NO.: 225-S 48 CTNNB1TTTCACTAACCTGGTAAAAGAGGAT SEQ ID NO.: 291-S 49 CTNNB1TTTACCAGGTTAGTGAAACGCAGAC SEQ ID NO.: 301-AS 50 CTNNB1TTTTACCAGGTTAGTGAAACGCAGA SEQ ID NO.: 302-AS 51 CTNNB1TTTTTTTTGTGGGTGTAATAGTGAC SEQ ID NO.: 321-S 52 CTNNB1TTTTTTTGTGGGTGTAATAGTGACA SEQ ID NO.: 322-S 53 CTNNB1TTTTTTGTGGGTGTAATAGTGACAT SEQ ID NO.: 323-S 54 CTNNB1TTTTTGTGGGTGTAATAGTGACATT SEQ ID NO.: 324-S 55 CTNNB1TTTTGTGGGTGTAATAGTGACATTT SEQ ID NO.: 325-S 56 CTNNB1_FWDTCCCACTGTACCTCTGTTATCCA SEQ ID NO.: 57 CTNNB1_REVTGGTCCTCGTCATTTAGCAGTTT SEQ ID NO.: 58

Referring to FIG. 4, T7EI assay revealed significant improvement of thetargeting efficiency by M537R and F870L mutations. First, M537R, F870Lor the double-mutant (M537R/F870L) enabled efficient cleavage at all 15sites with TTTT PAM, where 11 of 15 have no detectable cleavage byWT-Cpf1. For other sites with canonical TTTV PAM, these variantsmaintained or improved the targeting efficiency. The benefit isparticularly significant at those low-activity sites, such as CTNNB1111-s (3-fold improvement over WT). Among these variants, thedouble-mutant (M537R/F870) has the most consistent improvement oftargeting efficiency across all tested sites, where the singly mutantsexhibited more site-dependent variations, such as F870L at 323-S (noactivity, same as WT). Overall, the described invention exhibits vastlysuperior on-target potency than the WT-Cpf1.

Example 5 High-Throughput Measurement of the DNA Cleavage Activity ofAsCas12a Variants at TTTT PAM Site in E. coli

The following Example demonstrates the robustness of our novelhigh-throughput screening strategy to directly measure the cleavageactivity of thousands AsCas12a variants at TTTT PAM site in thebacterial-based activity assay (FIG. 5). FIG. 5A-F shows exemplaryhigh-throughput phenotype measurement of AsCas12a point mutations bydeep-scanning mutagenesis. A library encompassing every possible singlepoint mutation of AsCas12a in the targeted region (499-640 and 840-913)was generated in the context of WT-AsCas12a or M537R/F870L-AsCas12a. Therelative survival rate of each variant over the reference protein in anE. coil-based activity assay was determined by deep-sequencing. Thephenotype of individual point mutations was measured under multipleselection stringencies in the context of M537R/F870L (condition 1 and2), and a third condition in the WT-AsCas12a background (condition 3).As shown in FIG. 5A-C, the phenotype scores (i.e. natural logarithm ofrelative survival rate) of variants are positively correlated underdifferent conditions (ρ˜0.7), demonstrating the consistency andreproducibility of this approach. As the positive control, only theM537R and F870L/I, but not any other substitutions at these positions,survived more than the WT-AsCas12a (FIG. 5D). Conversely, mutating R537or L870 on the M537R/F870L-AsCas12a ubiquitously reduced the survivalrate (FIG. 5E). These results demonstrated that the phenotype scoremeasured by the bacterial screening reflects the DNA cleavage activityof previously characterized AsCas12a variants at TTTT PAM.

To further validate the result of our bacterial screen, we studied fourpoint mutations of AsCas12a with greater survival rate than thereference under all three conditions (L505K, S510L, P569D and P599G,FIG. 5F), Of note, P599G has been shown to enhance the cleavage activityof AsCas12a at TTCC PAM. The other three point mutations have not beencharacterized by any published studies so far. We therefore measured thesurvival rate of E. coli cells transformed with plasmids expressingindividual AsCas12a variant under selection. Compared to WT-AsCas12a,all selected point mutations increased the survival rate when targetinga TTTT-PAM site (FIG. 6). Unexpectedly, the benefits of these pointmutations held true even in the context of M537R/F870L-AsCas12a, wherethe survival rate was further elevated (FIG. 7). Collectively, theseresults demonstrated that our high-throughput screening can accuratelypredict the phenotype of uncharacterized AsCas12a variants.

The phenotype scores of 3,194 AsCas12a variants with single pointmutation covered by the screening with sufficient sequencing count arelisted in Table 3. Overall, ˜60% exhibited some benefits (i.e. phenotypescore>0) in one of the three condition.

TABLE 3 Summary of selected variants in different backgroundconditions.¹ Consistent Enrich. Stand. Enrich. Stand. Enrich. Stand. Anypositive positive Mutant Score (1) Error (1) Score (2) Error (2) Score(3) Error (3) enrichment? enrichment? R499C 0.14 0.08 0.60 0.07 0.060.10 Yes Yes R499L 0.08 0.05 0.05 0.06 0.09 0.15 Yes Yes R499K 0.54 0.060.23 0.06 0.38 0.10 Yes Yes R499A −0.29 0.05 0.27 0.04 0.22 0.07 Yes NoR499N 0.39 0.11 −0.49 0.15 −0.13 0.29 Yes No R499D 0.03 0.09 −1.01 0.12−0.51 0.44 Yes No R499Q −0.10 0.08 0.42 0.07 0.19 0.20 Yes No R499E−0.23 0.06 0.50 0.05 −0.10 0.10 Yes No R499G −0.11 0.03 0.12 0.03 0.140.06 Yes No R499H −0.45 0.11 0.50 0.09 NA NA Yes No R499I −0.14 0.100.09 0.09 −0.18 0.13 Yes No R499M −0.44 0.09 0.50 0.07 0.27 0.21 Yes NoR499F 0.01 0.15 −0.31 0.17 NA NA Yes No R499P −0.56 0.08 −0.06 0.07−0.70 0.26 No No R499S −0.14 0.05 0.02 0.05 −0.04 0.06 Yes No R499*−0.76 0.10 −0.61 0.10 −1.06 0.18 No No R499T −0.01 0.07 0.56 0.06 0.230.16 Yes No R499W −0.27 0.05 0.11 0.05 −0.53 0.11 Yes No R499V 0.09 0.050.09 0.05 −0.10 0.14 Yes No L500M 0.01 0.05 0.30 0.05 0.28 0.12 Yes YesL500A −0.27 0.04 0.21 0.03 −0.15 0.07 Yes No L500R −0.35 0.03 −0.40 0.03−0.47 0.05 No No L500N −1.11 0.14 −0.60 0.12 −0.50 0.16 No No L500D−1.03 0.08 −1.98 0.13 −1.26 0.19 No No L500C −0.19 0.05 0.26 0.05 0.000.10 Yes No L500Q −0.67 0.06 −0.63 0.06 −0.13 0.07 No No L500E −1.000.06 −1.22 0.07 −1.29 0.07 No No L500G −0.72 0.03 −0.79 0.03 −0.84 0.03No No L500H −0.30 0.09 0.01 0.08 0.01 0.22 Yes No L500I −0.31 0.09 0.070.08 0.26 0.15 Yes No L500K −1.21 0.09 −0.32 0.07 −0.28 0.07 No No L500F−0.19 0.08 −0.33 0.09 0.09 0.21 Yes No L500P −0.79 0.05 −0.80 0.05 −0.910.07 No No L500S −0.84 0.04 −0.43 0.04 −0.37 0.07 No No L500* −1.39 0.09−1.60 0.11 −1.66 0.16 No No L500T −0.19 0.05 −0.37 0.06 −0.17 0.06 No NoL500W −0.25 0.04 −0.29 0.04 0.01 0.11 Yes No L500Y −0.01 0.09 0.04 0.090.05 0.22 Yes No L500V −0.13 0.03 0.11 0.03 −0.03 0.06 Yes No T501L 0.180.04 0.32 0.04 0.12 0.07 Yes Yes T501M 0.32 0.07 0.35 0.07 0.17 0.29 YesYes T501V 0.22 0.06 0.29 0.06 0.13 0.20 Yes Yes T501A −0.06 0.03 −0.130.03 −0.07 0.10 No No T501R 0.01 0.03 −0.16 0.03 0.54 0.07 Yes No T501N0.69 0.14 0.16 0.15 NA NA Yes No T501D 0.12 0.12 −0.12 0.13 NA NA Yes NoT501C 0.52 0.09 −0.40 0.11 0.31 0.26 Yes No T501Q −0.19 0.10 −0.04 0.10−0.24 0.31 No No T501E −0.39 0.09 0.05 0.08 −0.34 0.26 Yes No T501G 0.120.04 0.44 0.04 −0.03 0.09 Yes No T501I 0.13 0.07 0.05 0.07 −0.19 0.11Yes No T501K −0.30 0.09 0.21 0.08 0.55 0.24 Yes No T501F 0.08 0.08 −0.120.08 −0.12 0.14 Yes No T501P −0.29 0.07 0.14 0.06 −0.39 0.13 Yes NoT501S 0.06 0.05 0.14 0.05 −0.10 0.09 Yes No T501* −1.18 0.12 −0.58 0.10−0.85 0.21 No No T501W −0.02 0.07 0.26 0.06 −0.30 0.09 Yes No T501Y−0.08 0.12 0.11 0.12 −0.29 0.29 Yes No G502R 0.14 0.05 0.28 0.05 0.980.07 Yes Yes G502E 0.42 0.08 0.17 0.08 0.19 0.30 Yes Yes G502L 0.45 0.070.10 0.07 0.00 0.11 Yes Yes G502S 0.08 0.06 0.23 0.06 0.16 0.12 Yes YesG502W 0.14 0.07 0.65 0.06 0.36 0.14 Yes Yes G502V 0.21 0.05 0.21 0.050.13 0.07 Yes Yes G502A −0.05 0.05 −0.34 0.05 0.08 0.07 Yes No G502D−0.14 0.08 0.37 0.07 0.10 0.15 Yes No G502C −0.17 0.08 0.23 0.08 −0.040.13 Yes No G502Q 0.78 0.11 0.02 0.13 −0.06 0.55 Yes No G502H 0.61 0.150.01 0.17 NA NA Yes No G502M −0.34 0.13 0.35 0.11 NA NA Yes No G502F NANA NA NA 0.08 0.37 Yes No G502P −0.41 0.14 0.01 0.13 NA NA Yes No G502*−1.16 0.15 −0.82 0.13 NA NA Yes No G502T 0.69 0.10 −0.23 0.13 0.24 0.35Yes No I503A −0.76 0.09 0.32 0.07 −0.16 0.21 Yes No I503R −0.86 0.07−0.67 0.07 −0.51 0.17 No No I503N −0.03 0.09 −0.30 0.10 −0.26 0.17 No NoI503D −0.50 0.13 −0.57 0.13 NA NA Yes No I503C −0.24 0.11 0.84 0.09 0.000.33 Yes No I503E NA NA NA NA −0.71 0.36 Yes No I503G −0.69 0.06 −0.300.05 −0.53 0.15 No No I503L 0.13 0.06 −0.46 0.07 −0.31 0.15 Yes No I503K0.03 0.12 −0.51 0.15 NA NA Yes No I503M −0.08 0.10 −0.17 0.10 −0.13 0.27No No I503F −0.17 0.09 −0.17 0.09 −0.25 0.19 No No I503S −0.45 0.07−0.27 0.07 −0.23 0.19 No No I503T −0.23 0.08 0.33 0.07 0.20 0.14 Yes NoI503W −1.21 0.11 −0.88 0.10 −0.61 0.28 No No I503V −0.09 0.05 0.34 0.04−0.03 0.09 Yes No K504A −0.15 0.07 −0.33 0.07 −0.30 0.20 No No K504R−0.05 0.03 0.04 0.03 −0.08 0.06 Yes No K504N 0.01 0.03 −0.54 0.04 −0.500.04 Yes No K504C −0.46 0.13 0.02 0.12 NA NA Yes No K504Q −0.16 0.05−0.25 0.05 −0.22 0.07 No No K504E −0.03 0.05 −0.32 0.06 −0.65 0.11 No NoK504G −0.84 0.05 −0.46 0.05 −0.52 0.15 No No K504H −0.59 0.13 −0.01 0.11−0.51 0.16 No No K504I −0.07 0.08 0.12 0.07 −0.31 0.14 Yes No K504L−0.39 0.05 −0.05 0.05 −0.10 0.14 No No K504M −0.32 0.08 −0.06 0.08 −0.060.10 No No K504F −0.71 0.14 −0.24 0.12 NA NA Yes No K504S −0.50 0.08−0.45 0.08 −0.19 0.23 No No K504* −0.43 0.09 −0.59 0.10 −0.76 0.15 No NoK504T −0.22 0.08 0.08 0.07 −0.15 0.14 Yes No K504W −0.48 0.08 0.09 0.07−0.53 0.24 Yes No K504V −0.48 0.05 −0.27 0.05 −0.36 0.12 No No L505A0.61 0.05 0.50 0.05 0.47 0.13 Yes Yes L505R 0.40 0.03 0.56 0.03 0.810.05 Yes Yes L505Q 0.30 0.07 0.82 0.07 0.16 0.11 Yes Yes L505E 0.18 0.060.03 0.07 0.14 0.21 Yes Yes L505G 0.18 0.03 0.72 0.03 0.40 0.05 Yes YesL505H 0.33 0.11 0.83 0.10 0.34 0.32 Yes Yes L505K 0.02 0.10 1.00 0.081.00 0.13 Yes Yes L505S 0.13 0.06 0.32 0.06 0.60 0.07 Yes Yes L505N−0.53 0.17 0.27 0.14 NA NA Yes No L505D −0.28 0.11 0.59 0.10 0.10 0.49Yes No L505C 0.26 0.07 −0.14 0.08 0.47 0.27 Yes No L505M −0.05 0.09−0.06 0.09 −0.18 0.18 No No L505F −0.97 0.15 0.28 0.11 −0.33 0.41 Yes NoL505P −0.55 0.07 −0.09 0.06 −0.09 0.11 No No L505T −0.23 0.09 0.44 0.080.59 0.17 Yes No L505W −0.03 0.06 0.28 0.05 −0.19 0.17 Yes No L505V−0.03 0.04 0.15 0.04 0.45 0.11 Yes No E506A 0.33 0.03 0.47 0.03 0.700.05 Yes Yes E506R 0.46 0.03 0.81 0.03 1.20 0.04 Yes Yes E506N 0.24 0.090.53 0.09 0.50 0.29 Yes Yes E506C 0.20 0.06 0.29 0.06 0.49 0.19 Yes YesE506Q 0.09 0.07 0.74 0.06 0.33 0.15 Yes Yes E506G 0.24 0.02 0.39 0.020.53 0.03 Yes Yes E506H 0.35 0.09 0.10 0.10 0.98 0.31 Yes Yes E506I 0.470.09 0.45 0.09 0.69 0.13 Yes Yes E506L 0.30 0.04 0.61 0.04 0.56 0.07 YesYes E506K 0.18 0.06 0.59 0.05 1.08 0.08 Yes Yes E506M 0.53 0.06 0.280.07 0.47 0.17 Yes Yes E506S 0.01 0.05 0.47 0.04 0.50 0.06 Yes Yes E506T0.39 0.06 0.12 0.07 0.90 0.23 Yes Yes E506Y 0.16 0.09 0.29 0.09 0.430.25 Yes Yes E506V 0.45 0.03 0.51 0.03 0.75 0.05 Yes Yes E506D 0.19 0.04−0.30 0.05 −0.08 0.08 Yes No E506F −0.21 0.10 0.47 0.08 0.20 0.25 Yes NoE506P −0.07 0.08 0.37 0.07 0.52 0.26 Yes No E506* −0.79 0.08 −0.79 0.08−1.11 0.07 No No E506W 0.43 0.04 0.10 0.04 −0.07 0.05 Yes No M507A −0.470.06 −0.34 0.06 −0.32 0.19 No No M507R −0.42 0.02 −0.58 0.03 −0.64 0.04No No M507C −0.41 0.09 −0.31 0.09 0.15 0.28 Yes No M507Q −0.68 0.07−0.56 0.07 −0.44 0.17 No No M507E NA NA NA NA −1.23 0.19 Yes No M507G−0.98 0.05 −0.71 0.05 −1.24 0.12 No No M507H −0.65 0.12 0.68 0.09 −0.860.28 Yes No M507I −0.06 0.04 −0.12 0.04 0.01 0.07 Yes No M507L −0.200.03 −0.35 0.03 −0.16 0.03 No No M507K −1.42 0.13 −0.06 0.09 −1.00 0.19No No M507F 0.05 0.09 −0.78 0.12 −0.05 0.36 Yes No M507P −2.08 0.12−2.19 0.13 −1.58 0.18 No No M507S −1.03 0.09 −0.34 0.07 −0.46 0.18 No NoM507* −1.68 0.15 −0.72 0.11 NA NA Yes No M507T −0.11 0.06 −0.30 0.07−0.03 0.13 No No M507W −0.94 0.08 −0.59 0.07 −0.82 0.18 No No M507V−0.17 0.03 0.06 0.03 −0.28 0.06 Yes No E508A 0.36 0.03 0.20 0.03 0.180.06 Yes Yes E508R 0.54 0.03 0.80 0.03 0.82 0.06 Yes Yes E508Q 0.25 0.060.11 0.07 0.51 0.13 Yes Yes E508G 0.16 0.02 0.17 0.02 0.22 0.04 Yes YesE508L 0.03 0.04 0.10 0.04 0.26 0.11 Yes Yes E508K 0.20 0.06 0.49 0.060.66 0.08 Yes Yes E508M 0.25 0.07 0.57 0.06 0.54 0.11 Yes Yes E508F 0.270.08 0.19 0.08 0.39 0.29 Yes Yes E508S 0.31 0.05 0.62 0.04 0.34 0.06 YesYes E508T 0.19 0.06 0.73 0.06 0.55 0.10 Yes Yes E508Y 0.35 0.09 0.190.10 0.21 0.27 Yes Yes E508V 0.16 0.03 0.22 0.03 0.34 0.05 Yes Yes E508N−0.08 0.11 −0.10 0.11 0.55 0.20 Yes No E508D 0.09 0.04 −0.15 0.04 −0.160.06 Yes No E508C −0.15 0.08 0.18 0.07 0.02 0.24 Yes No E508H −0.25 0.110.57 0.09 0.65 0.24 Yes No E508I 0.29 0.09 −0.12 0.10 0.54 0.15 Yes NoE508P −0.52 0.08 −0.67 0.08 −0.63 0.20 No No E508* −0.63 0.06 −0.82 0.07−0.79 0.09 No No E508W −0.29 0.05 0.45 0.05 0.07 0.09 Yes No P509R 0.230.03 0.27 0.03 1.04 0.05 Yes Yes P509K 0.01 0.07 0.12 0.07 1.29 0.13 YesYes P509M 0.34 0.07 0.34 0.07 0.10 0.22 Yes Yes P509S 0.10 0.04 0.140.04 0.14 0.10 Yes Yes P509W 0.04 0.05 0.33 0.05 0.61 0.18 Yes Yes P509Y0.18 0.09 0.20 0.09 0.53 0.30 Yes Yes P509A 0.45 0.04 −0.19 0.04 −0.040.05 Yes No P509N 0.18 0.09 −0.33 0.11 0.63 0.29 Yes No P509D −0.12 0.08−0.12 0.08 −0.01 0.21 No No P509C 0.00 0.06 −0.37 0.07 0.05 0.20 Yes NoP509Q 0.08 0.05 −0.10 0.06 0.36 0.08 Yes No P509E −0.27 0.06 0.33 0.050.38 0.17 Yes No P509G −0.06 0.03 0.19 0.03 0.00 0.05 Yes No P509H 0.090.03 −0.31 0.03 −0.19 0.05 Yes No P509I −0.10 0.09 −0.34 0.10 −0.56 0.28No No P509L 0.11 0.04 0.17 0.04 −0.17 0.05 Yes No P509F −0.12 0.09 0.240.08 0.52 0.11 Yes No P509* −2.22 0.15 −0.93 0.09 NA NA Yes No P509T0.10 0.06 0.58 0.05 −0.04 0.11 Yes No P509V 0.09 0.04 0.07 0.04 −0.450.08 Yes No S510G 0.18 0.03 0.49 0.03 0.21 0.06 Yes Yes S510L 0.57 0.060.72 0.06 0.74 0.09 Yes Yes S510A 0.29 0.06 −0.25 0.07 0.04 0.22 Yes NoS510R −0.38 0.03 −0.43 0.03 −0.53 0.04 No No S510N −0.36 0.09 −0.11 0.090.06 0.17 Yes No S510D −1.40 0.15 −1.36 0.15 NA NA Yes No S510C −0.140.06 0.03 0.06 0.28 0.14 Yes No S510E −0.71 0.09 −1.10 0.11 −1.07 0.25No No S510I 0.02 0.05 −0.39 0.06 −0.76 0.03 Yes No S510M −1.15 0.14−0.15 0.10 0.36 0.23 Yes No S510F −0.15 0.12 0.48 0.11 NA NA Yes NoS510* −1.11 0.13 −1.36 0.15 NA NA Yes No S510T 0.18 0.08 0.64 0.08 −0.170.16 Yes No S510W −0.06 0.06 −0.94 0.08 −0.37 0.17 No No S510V −0.050.05 0.44 0.05 0.44 0.18 Yes No L511A 0.07 0.10 −0.06 0.11 −0.31 0.15Yes No L511R 0.00 0.03 −0.21 0.04 0.09 0.06 Yes No L511Q −0.72 0.15 0.450.11 NA NA Yes No L511E −0.31 0.12 −0.25 0.13 −0.81 0.34 No No L511G−0.42 0.07 0.08 0.06 −0.02 0.27 Yes No L511K 0.26 0.13 0.44 0.12 NA NAYes No L511M −0.67 0.11 0.33 0.08 0.25 0.20 Yes No L511P 0.04 0.08 0.000.09 0.26 0.13 Yes No L511S −0.12 0.12 −0.37 0.13 0.02 0.33 Yes No L511W0.61 0.08 0.02 0.09 −0.40 0.38 Yes No L511V −0.26 0.06 0.38 0.06 −0.020.16 Yes No S512R 0.07 0.07 0.34 0.07 0.07 0.19 Yes Yes S512A 0.11 0.05−0.15 0.06 −0.16 0.06 Yes No S512N −0.12 0.19 0.58 0.16 NA NA Yes NoS512D −0.14 0.12 −0.33 0.13 0.47 0.39 Yes No S512C 0.06 0.08 0.61 0.08−0.18 0.19 Yes No S512E 0.44 0.10 −0.11 0.12 0.21 0.36 Yes No S512G−0.15 0.05 −0.15 0.05 0.10 0.12 Yes No S512L −0.16 0.10 0.53 0.09 −0.210.20 Yes No S512K 0.65 0.15 0.57 0.15 NA NA Yes No S512M 0.28 0.14 0.240.15 NA NA Yes No S512F −0.28 0.08 −0.16 0.08 −0.32 0.13 No No S512P−0.22 0.08 −0.09 0.08 −0.16 0.13 No No S512T −0.17 0.09 0.05 0.08 −0.070.15 Yes No S512W −0.13 0.10 0.13 0.10 NA NA Yes No S512Y −0.26 0.13−0.68 0.15 NA NA Yes No S512V −0.38 0.08 −0.54 0.09 −0.64 0.19 No NoF513L 0.53 0.04 0.63 0.04 0.41 0.07 Yes Yes F513W 0.04 0.06 0.35 0.060.28 0.13 Yes Yes F513A −1.58 0.11 0.20 0.06 −0.49 0.25 Yes No F513R−1.56 0.09 −1.47 0.09 −0.89 0.06 No No F513D −1.02 0.15 0.12 0.11 NA NAYes No F513C −0.83 0.10 −0.27 0.09 −0.27 0.14 No No F513Q 0.00 0.13 0.580.12 NA NA Yes No F513E −0.37 0.09 0.33 0.08 −0.38 0.38 Yes No F513G−1.33 0.06 −0.80 0.05 −1.00 0.13 No No F513I 0.78 0.10 0.21 0.11 NA NAYes No F513M −0.36 0.12 0.73 0.10 0.49 0.23 Yes No F513S −0.59 0.07−0.47 0.07 −0.61 0.10 No No F513T NA NA NA NA −0.35 0.19 Yes No F513Y−0.10 0.10 0.26 0.09 0.44 0.12 Yes No F513V −0.53 0.06 −0.27 0.06 0.060.19 Yes No Y514A −0.28 0.05 −0.43 0.05 −0.73 0.13 No No Y514R −0.230.04 −0.47 0.05 −0.86 0.14 No No Y514N −0.17 0.09 0.07 0.08 −0.53 0.17Yes No Y514D −1.11 0.10 −1.20 0.11 −1.22 0.16 No No Y514C −0.39 0.05−0.35 0.05 −0.55 0.09 No No Y514Q −1.35 0.13 −0.62 0.10 −1.01 0.29 No NoY514E −1.08 0.08 −0.93 0.08 −1.50 0.19 No No Y514G −0.76 0.04 −0.49 0.04−1.06 0.11 No No Y514H 0.39 0.06 0.29 0.06 −0.31 0.12 Yes No Y514I −0.140.09 −0.84 0.11 NA NA Yes No Y514L −0.37 0.05 −0.57 0.05 −0.73 0.10 NoNo Y514K −0.52 0.09 −1.28 0.12 NA NA Yes No Y514M −0.50 0.07 −0.38 0.07−0.59 0.19 No No Y514F −0.29 0.07 0.14 0.06 −0.35 0.11 Yes No Y514S−0.33 0.05 −0.32 0.05 −0.86 0.11 No No Y514* −0.91 0.08 −1.20 0.10 −1.180.13 No No Y514T −1.01 0.09 −1.31 0.10 −0.98 0.18 No No Y514W 0.17 0.050.46 0.05 −0.09 0.23 Yes No Y514V −0.45 0.04 −0.41 0.04 −0.92 0.06 No NoN515A 0.60 0.06 0.82 0.06 0.12 0.11 Yes Yes N515R 0.63 0.06 0.82 0.050.19 0.11 Yes Yes N515I 0.23 0.07 0.30 0.07 0.16 0.14 Yes Yes N515L 0.330.06 0.53 0.06 0.07 0.20 Yes Yes N515T 0.41 0.08 0.14 0.08 0.29 0.16 YesYes N515V 0.50 0.05 0.49 0.05 0.45 0.09 Yes Yes N515D 0.05 0.06 −0.170.06 −0.30 0.10 Yes No N515C 0.59 0.12 0.62 0.12 −0.02 0.30 Yes No N515Q0.46 0.11 0.69 0.11 NA NA Yes No N515E 0.47 0.07 0.56 0.07 −0.14 0.19Yes No N515G −0.10 0.05 0.37 0.04 −0.13 0.07 Yes No N515H 0.09 0.10−0.59 0.13 −0.25 0.21 Yes No N515K 0.07 0.08 0.16 0.08 −0.13 0.16 Yes NoN515M 0.73 0.09 0.45 0.10 −0.01 0.12 Yes No N515F 0.49 0.12 0.48 0.12 NANA Yes No N515P −1.10 0.16 −0.13 0.12 −0.32 0.38 No No N515S 0.08 0.05−0.15 0.05 0.19 0.08 Yes No N515W 0.79 0.08 0.42 0.09 −0.60 0.31 Yes NoN515Y −0.30 0.12 −0.55 0.13 0.03 0.22 Yes No K516R 0.01 0.03 0.26 0.030.40 0.06 Yes Yes K516A 0.25 0.05 0.13 0.06 −0.33 0.21 Yes No K516N 0.370.09 0.01 0.11 −0.01 0.16 Yes No K516D NA NA NA NA −0.48 0.31 Yes NoK516C −1.37 0.13 0.14 0.08 −0.36 0.16 Yes No K516Q 0.08 0.07 −0.76 0.10−0.08 0.16 Yes No K516E −0.23 0.04 −0.32 0.05 −0.33 0.09 No No K516G−0.34 0.03 −0.01 0.03 −0.34 0.05 No No K516I −0.29 0.14 −0.03 0.14 NA NAYes No K516L −0.29 0.06 −0.06 0.06 −0.56 0.18 No No K516M −0.29 0.08−0.21 0.08 −0.31 0.17 No No K516P −0.37 0.10 −0.96 0.12 NA NA Yes NoK516S −0.34 0.06 0.23 0.06 −0.22 0.21 Yes No K516* −0.66 0.09 −0.86 0.10−1.08 0.19 No No K516T −0.18 0.04 −0.43 0.04 −0.09 0.06 No No K516W−0.42 0.06 −0.55 0.07 −1.18 0.16 No No K516V −0.47 0.05 −0.20 0.05 −1.040.11 No No A517R −2.02 0.07 −1.56 0.06 −1.46 0.07 No No A517D −1.35 0.11−0.85 0.09 −1.19 0.18 No No A517C −0.35 0.08 0.68 0.07 0.14 0.17 Yes NoA517E −1.34 0.09 −1.16 0.09 −1.86 0.16 No No A517G −0.69 0.03 −0.65 0.03−0.77 0.04 No No A517I 0.77 0.10 −0.42 0.13 NA NA Yes No A517L −0.300.06 −0.44 0.06 −1.22 0.17 No No A517M −0.50 0.10 −0.24 0.10 −0.57 0.30No No A517F −0.50 0.12 −0.90 0.14 NA NA Yes No A517P −0.75 0.08 −1.060.09 −0.86 0.12 No No A517S −0.28 0.05 −0.24 0.05 −0.65 0.14 No No A517T−0.34 0.06 0.06 0.06 −0.42 0.11 Yes No A517W −1.22 0.07 −0.27 0.05 −1.430.16 No No A517Y 0.02 0.13 0.04 0.13 NA NA Yes No A517V −0.33 0.04 −0.140.04 −0.44 0.07 No No R518K 0.20 0.09 0.72 0.08 0.05 0.17 Yes Yes R518A−0.12 0.07 −0.28 0.07 −0.87 0.24 No No R518C −1.13 0.12 −0.21 0.09 NA NAYes No R518E −0.72 0.10 −1.08 0.12 NA NA Yes No R518G −1.34 0.05 −0.780.04 −0.87 0.08 No No R518L −1.49 0.12 −1.84 0.15 NA NA Yes No R518F−0.96 0.16 0.45 0.11 NA NA Yes No R518S −0.74 0.08 −1.20 0.09 −0.73 0.12No No R518* −0.33 0.08 −0.34 0.08 −0.69 0.13 No No R518T −0.50 0.10−0.37 0.10 −0.40 0.15 No No R518W −1.01 0.09 −1.13 0.10 −1.22 0.19 No NoR518V −0.44 0.06 −1.39 0.09 −1.43 0.09 No No N519A 0.64 0.06 1.07 0.06−0.06 0.25 Yes No N519R 0.47 0.05 0.46 0.05 −0.02 0.08 Yes No N519D 0.100.05 −0.13 0.05 −0.29 0.07 Yes No N519C 0.21 0.11 0.23 0.11 NA NA Yes NoN519Q 0.14 0.12 0.71 0.11 NA NA Yes No N519E −0.66 0.10 −0.38 0.09 −0.890.23 No No N519G 0.21 0.04 0.10 0.04 −0.54 0.08 Yes No N519H 0.10 0.09−0.67 0.11 NA NA Yes No N519I 0.29 0.09 0.18 0.09 −0.02 0.15 Yes NoN519L 0.28 0.06 0.47 0.06 −0.37 0.08 Yes No N519K −0.17 0.09 −0.35 0.10−0.46 0.19 No No N519M −0.19 0.10 0.36 0.09 −0.05 0.13 Yes No N519F 0.380.14 0.21 0.15 NA NA Yes No N519P −0.72 0.13 −0.23 0.11 NA NA Yes NoN519S 0.08 0.05 −0.03 0.05 0.02 0.12 Yes No N519T 0.24 0.08 0.01 0.09−0.37 0.16 Yes No N519W −0.02 0.08 0.38 0.08 −0.12 0.31 Yes No N519Y0.10 0.12 0.65 0.11 −0.09 0.22 Yes No N519V 0.12 0.05 0.14 0.05 −0.390.06 Yes No Y520A −1.19 0.12 −0.30 0.09 NA NA Yes No Y520R −0.37 0.08−1.30 0.11 −1.04 0.24 No No Y520N −0.05 0.09 −0.45 0.10 −0.62 0.14 No NoY520D −0.50 0.12 −0.04 0.10 NA NA Yes No Y520C −0.23 0.07 −0.24 0.07−0.52 0.12 No No Y520E −0.61 0.13 −0.85 0.15 NA NA Yes No Y520G −1.910.10 −1.08 0.08 −1.47 0.09 No No Y520H 0.12 0.07 −0.14 0.07 −0.34 0.10Yes No Y520F 0.19 0.13 −0.21 0.15 NA NA Yes No Y520S −0.73 0.11 −0.660.11 −1.13 0.18 No No Y520* −0.19 0.07 −0.42 0.08 −0.48 0.15 No No Y520W−0.38 0.10 0.06 0.09 −0.03 0.16 Yes No Y520V −1.45 0.13 −1.16 0.12 NA NAYes No A521D −0.72 0.13 −0.50 0.13 NA NA Yes No A521C 0.90 0.16 1.290.16 NA NA Yes No A521G −0.52 0.05 −0.73 0.06 −0.98 0.12 No No A521L−0.61 0.10 −0.13 0.09 −0.98 0.12 No No A521P −0.19 0.11 −0.78 0.14 NA NAYes No A521S 0.00 0.09 0.04 0.10 −0.51 0.27 Yes No A521T −0.23 0.07 0.100.06 −0.33 0.11 Yes No A521V −0.40 0.06 −0.88 0.07 −0.60 0.13 No NoT522L 1.10 0.05 0.92 0.05 0.28 0.10 Yes Yes T522M 0.92 0.07 0.81 0.080.53 0.21 Yes Yes T522V 0.16 0.05 0.28 0.05 0.04 0.07 Yes Yes T522A−0.01 0.05 −0.37 0.06 −0.55 0.08 No No T522R −0.17 0.04 0.31 0.04 −0.400.12 Yes No T522N 0.11 0.07 −1.14 0.11 −0.50 0.13 Yes No T522C 0.09 0.080.38 0.08 −0.41 0.38 Yes No T522Q 0.71 0.09 0.47 0.10 −0.51 0.36 Yes NoT522E −0.22 0.08 −1.54 0.13 −0.72 0.27 No No T522G −0.37 0.04 −0.18 0.04−0.85 0.08 No No T522I 0.23 0.07 −0.06 0.08 −0.11 0.17 Yes No T522K−0.16 0.11 −0.23 0.11 −0.67 0.26 No No T522F 0.08 0.13 0.34 0.12 NA NAYes No T522P −1.16 0.11 −1.52 0.14 −1.14 0.16 No No T522S 0.23 0.05−0.20 0.05 −0.38 0.10 Yes No T522W −0.26 0.07 −0.35 0.07 −0.90 0.13 NoNo K523R 0.17 0.02 0.37 0.02 0.00 0.03 Yes Yes K523A −0.19 0.03 0.150.03 −0.48 0.09 Yes No K523N −0.31 0.07 −0.48 0.07 −1.00 0.08 No NoK523D −1.14 0.07 −1.35 0.08 −1.21 0.09 No No K523C −0.22 0.05 0.07 0.04−0.81 0.12 Yes No K523Q −0.12 0.04 −0.22 0.04 0.01 0.06 Yes No K523E−0.57 0.04 −0.56 0.04 −0.86 0.06 No No K523G −0.16 0.02 0.09 0.02 −0.560.03 Yes No K523H −1.08 0.11 −0.05 0.08 −1.04 0.28 No No K523I −0.200.07 0.41 0.06 −1.23 0.21 Yes No K523L −0.78 0.04 −0.30 0.03 −0.70 0.07No No K523M −0.19 0.04 −0.18 0.04 −0.52 0.06 No No K523F −1.08 0.08−0.93 0.08 −1.27 0.09 No No K523P −1.41 0.08 −0.39 0.06 −1.21 0.15 No NoK523S 0.13 0.03 0.10 0.03 −0.36 0.07 Yes No K523* −1.40 0.07 −1.46 0.08−1.19 0.12 No No K523T −0.19 0.04 −0.19 0.04 −0.49 0.08 No No K523W−0.97 0.04 −0.87 0.04 −1.41 0.09 No No K523Y −1.25 0.10 −0.43 0.08 −1.360.19 No No K523V −0.57 0.03 −0.57 0.03 −1.12 0.08 No No K524A −0.88 0.09−0.32 0.08 −0.50 0.24 No No K524R −0.32 0.04 0.15 0.04 −0.28 0.09 Yes NoK524N 0.04 0.04 −0.48 0.05 −0.19 0.09 Yes No K524C 0.06 0.10 0.03 0.10NA NA Yes No K524Q −0.42 0.09 −0.54 0.10 −0.47 0.14 No No K524E −0.530.06 −0.11 0.06 −0.49 0.10 No No K524G −0.43 0.04 −0.19 0.04 −0.80 0.06No No K524L −0.25 0.07 0.32 0.06 −0.81 0.21 Yes No K524M −1.28 0.15−0.75 0.13 −0.69 0.15 No No K524P −0.70 0.12 −0.10 0.10 0.02 0.29 Yes NoK524S −0.67 0.08 −0.04 0.07 −0.73 0.12 No No K524* −0.92 0.11 −0.75 0.11−0.76 0.13 No No K524T −0.10 0.04 −0.26 0.04 −0.15 0.06 No No K524W−0.58 0.08 −1.54 0.12 −1.19 0.22 No No K524V −0.71 0.07 −0.01 0.06 −0.680.22 No No P525A −0.21 0.03 0.06 0.03 −0.29 0.07 Yes No P525R −0.30 0.02−0.10 0.02 −0.62 0.05 No No P525N −0.38 0.09 −0.35 0.10 −0.10 0.19 No NoP525D 0.42 0.05 −0.02 0.05 −0.30 0.08 Yes No P525C −0.28 0.05 −0.12 0.05−0.48 0.14 No No P525Q −0.21 0.06 −0.19 0.06 −0.35 0.13 No No P525E−0.22 0.04 0.24 0.04 −0.30 0.05 Yes No P525G −0.24 0.02 −0.17 0.02 −0.510.05 No No P525H 0.17 0.06 0.18 0.07 −0.24 0.12 Yes No P525I −0.48 0.090.06 0.08 0.19 0.22 Yes No P525L −0.37 0.03 0.25 0.03 −0.61 0.06 Yes NoP525K −0.90 0.08 −1.00 0.09 −0.57 0.17 No No P525M −0.55 0.06 −0.18 0.06−0.54 0.13 No No P525F −0.17 0.07 −0.61 0.08 −0.70 0.16 No No P525S−0.06 0.03 0.04 0.03 −0.42 0.08 Yes No P525* −1.61 0.09 −1.54 0.09 −1.560.08 No No P525T −0.25 0.05 −0.31 0.05 −0.45 0.11 No No P525W −0.25 0.04−0.04 0.03 −0.59 0.11 No No P525Y −1.08 0.10 −1.08 0.10 −0.77 0.08 No NoP525V −0.31 0.03 −0.08 0.03 −0.39 0.08 No No Y526A −0.28 0.07 0.17 0.06−0.89 0.19 Yes No Y526R −0.71 0.07 −0.17 0.06 −1.09 0.19 No No Y526N−0.64 0.11 −0.15 0.09 −0.25 0.17 No No Y526D −0.44 0.09 0.07 0.08 −0.690.15 Yes No Y526C 0.04 0.06 −0.03 0.06 −0.26 0.12 Yes No Y526Q −0.100.11 −0.64 0.13 NA NA Yes No Y526E −0.46 0.08 −0.87 0.10 −0.84 0.21 NoNo Y526G −0.22 0.05 −0.17 0.05 −0.65 0.16 No No Y526H −0.32 0.08 −0.250.08 −0.19 0.12 No No Y526I −0.40 0.13 −0.52 0.14 NA NA Yes No Y526L−0.53 0.06 −0.18 0.05 −0.73 0.12 No No Y526M −0.47 0.10 −0.40 0.10 −1.120.27 No No Y526F −0.02 0.07 −0.01 0.07 −0.44 0.13 No No Y526P −0.19 0.10−0.88 0.13 NA NA Yes No Y526S −0.57 0.06 −0.68 0.07 −0.68 0.12 No NoY526* −0.42 0.05 −0.44 0.06 −0.61 0.09 No No Y526T −0.01 0.09 −0.22 0.10−0.69 0.21 No No Y526W −1.02 0.10 −0.19 0.08 −0.32 0.13 No No Y526V−0.61 0.05 −0.14 0.05 −0.86 0.14 No No S527D 0.13 0.11 0.01 0.11 0.340.41 Yes Yes S527A 0.00 0.06 −0.37 0.07 −0.07 0.20 Yes No S527R −0.140.04 −0.73 0.05 −0.70 0.07 No No S527N −0.10 0.10 −0.43 0.11 0.01 0.19Yes No S527C −0.21 0.09 −0.35 0.09 −0.10 0.22 No No S527Q 0.09 0.12−0.39 0.14 NA NA Yes No S527E −0.49 0.10 0.57 0.08 0.03 0.16 Yes NoS527G −0.43 0.04 −0.21 0.04 −0.53 0.09 No No S527H 0.47 0.15 0.10 0.17NA NA Yes No S527I −1.06 0.16 −0.43 0.14 NA NA Yes No S527L −0.12 0.07−0.24 0.07 −0.44 0.27 No No S527K −0.09 0.11 −0.57 0.13 NA NA Yes NoS527P 0.07 0.10 −0.33 0.11 −0.47 0.36 Yes No S527T 0.01 0.09 −0.30 0.10−0.22 0.18 Yes No S527W 0.03 0.07 0.15 0.07 −0.50 0.23 Yes No S527Y 0.310.17 0.05 0.18 NA NA Yes No S527V −0.54 0.07 −0.52 0.07 −0.52 0.10 No NoV528D 0.01 0.07 0.27 0.07 0.12 0.12 Yes Yes V528L 0.55 0.03 0.59 0.030.31 0.07 Yes Yes V528M 0.19 0.05 0.02 0.05 0.13 0.10 Yes Yes V528A−0.09 0.04 0.29 0.04 −0.13 0.06 Yes No V528R −0.04 0.04 0.07 0.04 −0.030.07 Yes No V528N 0.01 0.11 −0.47 0.14 0.31 0.42 Yes No V528C 0.16 0.070.20 0.07 −0.06 0.24 Yes No V528Q −0.44 0.09 −0.29 0.09 0.07 0.11 Yes NoV528E −0.66 0.06 0.16 0.05 0.00 0.11 Yes No V528G −0.14 0.02 −0.19 0.02−0.19 0.03 No No V528H −0.62 0.15 1.01 0.11 NA NA Yes No V528I 0.16 0.08−0.09 0.09 0.07 0.11 Yes No V528K −0.14 0.08 0.36 0.08 0.13 0.14 Yes NoV528F −0.10 0.09 0.39 0.08 −0.59 0.21 Yes No V528P −0.10 0.08 −0.66 0.100.10 0.11 Yes No V528S −0.12 0.05 0.26 0.05 0.11 0.06 Yes No V528* −1.660.13 −1.20 0.12 NA NA Yes No V528T 0.29 0.07 0.00 0.07 0.18 0.17 Yes NoV528W −1.31 0.07 −1.47 0.08 −0.99 0.14 No No V528Y −0.77 0.13 −0.50 0.120.24 0.12 Yes No E529A −0.38 0.08 −0.18 0.08 0.04 0.15 Yes No E529R NANA NA NA −0.70 0.34 Yes No E529D 0.02 0.07 −0.61 0.08 0.10 0.15 Yes NoE529Q −0.43 0.14 −0.49 0.15 NA NA Yes No E529G −0.25 0.05 −0.16 0.05−0.61 0.11 No No E529L −0.45 0.11 −0.56 0.11 NA NA Yes No E529K 0.210.10 −0.28 0.11 −0.65 0.19 Yes No E529P 0.08 0.15 −0.30 0.17 NA NA YesNo E529S −0.18 0.11 −0.08 0.11 −0.20 0.45 No No E529* −0.22 0.11 −0.690.14 −0.58 0.17 No No E529W −0.19 0.13 −0.11 0.13 NA NA Yes No E529V−0.35 0.06 0.08 0.06 −0.48 0.10 Yes No K530A −0.96 0.10 −0.76 0.10 −0.900.14 No No K530R −0.56 0.04 −0.32 0.04 −0.59 0.08 No No K530N −0.78 0.15−0.73 0.16 NA NA Yes No K530C −0.10 0.10 −0.43 0.11 NA NA Yes No K530Q−0.22 0.05 −0.56 0.05 −0.60 0.10 No No K530E −0.48 0.05 −0.35 0.05 −0.850.09 No No K530G −1.18 0.07 −1.09 0.07 −1.15 0.14 No No K530L −1.74 0.09−1.04 0.07 −1.40 0.14 No No K530M −0.72 0.07 −0.42 0.07 −0.83 0.13 No NoK530F −1.17 0.15 −0.40 0.12 NA NA Yes No K530S −1.15 0.09 −0.65 0.08−1.15 0.13 No No K530* −0.63 0.07 −0.43 0.07 −0.68 0.09 No No K530T−0.51 0.07 −0.72 0.07 −0.68 0.10 No No K530W −1.73 0.12 −0.35 0.08 −1.350.20 No No K530V −0.98 0.08 −1.46 0.10 −1.55 0.23 No No F531A −1.39 0.13−1.72 0.15 NA NA Yes No F531R −1.90 0.12 −1.41 0.10 −1.75 0.23 No NoF531C −0.40 0.05 −0.81 0.06 −0.85 0.09 No No F531G −1.68 0.08 −1.22 0.07−1.58 0.14 No No F531I −0.43 0.09 −0.86 0.11 −0.62 0.11 No No F531L−0.35 0.05 −0.40 0.05 −0.55 0.07 No No F531S −0.56 0.07 −0.79 0.08 −0.980.11 No No F531W −1.68 0.13 −0.86 0.10 −0.27 0.21 No No F531Y −0.99 0.16−0.55 0.14 NA NA Yes No F531V −0.22 0.02 −0.64 0.03 −0.74 0.05 No NoK532A NA NA NA NA −0.93 0.19 Yes No K532R −0.63 0.05 −0.63 0.05 −0.560.10 No No K532N −0.39 0.11 −0.71 0.13 −0.45 0.12 No No K532Q −0.42 0.08−0.74 0.10 −0.96 0.11 No No K532E −0.49 0.05 −0.38 0.05 −0.64 0.12 No NoK532G −1.74 0.08 −1.15 0.07 −1.35 0.11 No No K532L −1.28 0.11 −1.26 0.11−1.36 0.21 No No K532M −0.48 0.09 −0.58 0.10 −0.65 0.09 No No K532S NANA NA NA −0.93 0.25 Yes No K532* −0.42 0.06 −0.27 0.06 −0.65 0.12 No NoK532T −0.37 0.06 −1.05 0.07 −0.52 0.10 No No K532V −1.99 0.11 −1.32 0.09−1.19 0.12 No No L533R −0.17 0.04 −0.41 0.04 −0.57 0.08 No No L533G−1.82 0.14 −1.54 0.13 NA NA Yes No L533M −0.36 0.12 −0.29 0.12 −0.120.17 No No L533P −0.18 0.07 −0.06 0.07 −0.28 0.12 No No L533V −1.49 0.12−0.64 0.09 −0.58 0.19 No No N534A −1.49 0.08 −1.30 0.08 −0.31 0.09 No NoN534R −1.13 0.05 −1.37 0.06 −0.52 0.04 No No N534D −1.07 0.08 −0.72 0.07−0.75 0.11 No No N534C −1.50 0.13 −1.27 0.12 0.19 0.20 Yes No N534Q NANA NA NA −0.67 0.24 Yes No N534E −2.05 0.10 −1.48 0.08 −1.32 0.13 No NoN534G −1.43 0.04 −0.96 0.04 −1.10 0.04 No No N534H −0.18 0.03 −0.50 0.04−0.20 0.06 No No N534I −0.54 0.08 0.00 0.07 −0.60 0.18 Yes No N534L−1.92 0.11 −1.79 0.11 −0.26 0.07 No No N534K −1.00 0.07 −0.74 0.07 −0.010.10 No No N534M −1.72 0.12 −0.90 0.09 0.88 0.19 Yes No N534F 0.11 0.10−1.16 0.16 0.16 0.30 Yes No N534P −0.33 0.07 −0.26 0.07 −1.27 0.16 No NoN534S −1.00 0.06 −0.72 0.06 0.22 0.05 Yes No N534* −1.20 0.09 −2.22 0.15−1.84 0.10 No No N534T −0.38 0.04 −0.40 0.04 0.49 0.06 Yes No N534W−1.54 0.08 −0.69 0.06 −0.29 0.07 No No N534Y −0.97 0.12 −0.47 0.10 −0.570.17 No No N534V −1.35 0.07 −1.17 0.06 −0.59 0.06 No No N534A NA NA NANA −0.33 0.09 Yes No N534R NA NA NA NA −0.55 0.04 Yes No N534D −0.110.09 −0.10 0.09 −0.69 0.10 No No N534C NA NA NA NA 0.16 0.19 Yes NoN534Q NA NA NA NA −0.72 0.24 Yes No N534E NA NA NA NA −1.34 0.13 Yes NoN534G NA NA NA NA −1.11 0.04 Yes No N534H NA NA NA NA −0.19 0.24 Yes NoN534I −0.14 0.19 −0.05 0.19 −0.61 0.19 No No N534L NA NA NA NA −0.220.07 Yes No N534K −0.47 0.14 −0.26 0.13 −0.06 0.09 No No N534M NA NA NANA 0.83 0.18 Yes No N534F NA NA NA NA 0.26 0.30 Yes No N534S −0.15 0.07−0.03 0.07 0.20 0.05 Yes No N534* NA NA NA NA −1.73 0.10 Yes No N534T NANA NA NA 0.91 0.15 Yes No N534W NA NA NA NA −0.32 0.07 Yes No N534Y−0.01 0.13 0.04 0.13 −0.53 0.16 Yes No N534V NA NA NA NA −0.57 0.06 YesNo F535A −1.29 0.08 −1.38 0.09 −1.53 0.15 No No F535R −2.11 0.10 −1.420.08 −1.73 0.13 No No F535C −0.48 0.06 −0.52 0.06 −0.79 0.05 No No F535E−2.40 0.14 −1.29 0.09 −1.54 0.09 No No F535G −1.68 0.06 −1.41 0.05 −1.600.04 No No F535I −0.99 0.13 −0.63 0.12 NA NA Yes No F535L −0.97 0.06−0.52 0.05 −0.92 0.05 No No F535M −0.78 0.11 −0.99 0.13 −0.68 0.24 No NoF535S −0.91 0.06 −0.54 0.06 −1.11 0.09 No No F535W −2.13 0.11 −1.41 0.09−1.73 0.15 No No F535Y −0.65 0.10 −0.50 0.10 −0.62 0.16 No No F535V−0.87 0.04 −1.00 0.05 −0.98 0.04 No No F535A NA NA NA NA −1.47 0.15 YesNo F535R NA NA NA NA −1.74 0.12 Yes No F535C −0.09 0.06 0.09 0.05 −0.540.06 Yes No F535E NA NA NA NA −1.65 0.09 Yes No F535G NA NA NA NA −1.930.04 Yes No F535L −0.04 0.06 0.03 0.06 −0.69 0.05 Yes No F535M NA NA NANA −0.67 0.21 Yes No F535S −0.14 0.08 0.00 0.08 −1.14 0.09 No No F535WNA NA NA NA −1.68 0.14 Yes No F535Y −0.19 0.12 −0.06 0.12 −0.53 0.17 NoNo F535V −0.05 0.05 −0.01 0.05 −0.76 0.06 No No Q536A NA NA NA NA −0.020.19 Yes No Q536R 0.02 0.08 −0.02 0.09 0.08 0.05 Yes No Q536D NA NA NANA 0.23 0.28 Yes No Q536C NA NA NA NA −1.02 0.28 Yes No Q536E 0.00 0.160.02 0.16 −0.66 0.08 Yes No Q536G NA NA NA NA 1.22 0.05 Yes No Q536H−0.05 0.19 −0.05 0.19 −0.34 0.22 No No Q536L −0.10 0.15 −0.11 0.15 −1.090.14 No No Q536K 0.56 0.09 −0.36 0.11 −0.09 0.13 Yes No Q536S NA NA NANA 0.60 0.09 Yes No Q536* 0.15 0.09 0.03 0.10 −0.74 0.10 Yes No Q536V NANA NA NA −1.88 0.09 Yes No R537A −1.11 0.10 −1.99 0.15 NA NA Yes NoR537Q −0.56 0.13 −1.08 0.16 NA NA Yes No R537E −1.40 0.13 −1.33 0.13 NANA Yes No R537G −1.10 0.05 −1.07 0.05 NA NA Yes No R537L −1.50 0.13−1.38 0.12 NA NA Yes No R537K −0.58 0.09 −0.81 0.10 NA NA Yes No R537M−1.57 0.02 −1.19 0.02 NA NA Yes No R537S −1.30 0.11 −0.65 0.08 NA NA YesNo R537W −1.42 0.10 −1.60 0.11 NA NA Yes No R537V −1.52 0.11 −1.85 0.12NA NA Yes No M537A NA NA NA NA −1.52 0.24 Yes No M537R NA NA NA NA 1.700.04 Yes No M537G NA NA NA NA −1.55 0.08 Yes No M537I NA NA NA NA −0.010.15 Yes No M537L NA NA NA NA −0.09 0.13 Yes No M537K NA NA NA NA 0.190.14 Yes No M537T NA NA NA NA −0.29 0.13 Yes No M537W NA NA NA NA −1.300.19 Yes No M537V NA NA NA NA −0.62 0.10 Yes No P538A NA NA NA NA −1.190.06 Yes No P538R NA NA NA NA −1.72 0.08 Yes No P538Q NA NA NA NA −1.460.11 Yes No P538E NA NA NA NA −1.79 0.22 Yes No P538G NA NA NA NA −1.670.09 Yes No P538H −0.20 0.17 −0.19 0.17 NA NA Yes No P538L −0.23 0.12−0.09 0.11 −1.49 0.05 No No P538S 0.19 0.12 0.18 0.12 −1.47 0.11 Yes NoP538T −0.08 0.06 0.13 0.06 −0.38 0.06 Yes No P538W NA NA NA NA −1.820.17 Yes No P538V NA NA NA NA −0.71 0.14 Yes No T539A 0.08 0.07 −0.030.08 −1.10 0.06 Yes No T539R NA NA NA NA −1.51 0.10 Yes No T539N 0.160.14 0.06 0.15 −1.00 0.17 Yes No T539C NA NA NA NA −1.11 0.14 Yes NoT539Q NA NA NA NA −1.19 0.20 Yes No T539E NA NA NA NA −1.61 0.12 Yes NoT539G NA NA NA NA −1.63 0.05 Yes No T539I −0.20 0.13 0.00 0.13 −1.040.16 No No T539L NA NA NA NA −2.07 0.13 Yes No T539K NA NA NA NA −1.230.26 Yes No T539M NA NA NA NA −1.64 0.18 Yes No T539P −0.33 0.14 −0.400.14 −1.51 0.14 No No T539S −0.03 0.05 −0.17 0.05 −0.14 0.04 No No T539VNA NA NA NA −1.65 0.11 Yes No L540R −0.17 0.15 −0.30 0.15 −1.27 0.14 NoNo L540Q 0.12 0.08 0.13 0.08 −0.05 0.08 Yes No L540G NA NA NA NA −1.830.13 Yes No L540M 0.04 0.08 −0.02 0.09 −0.13 0.07 Yes No L540P 0.05 0.090.09 0.09 −0.40 0.13 Yes No L540V NA NA NA NA −1.58 0.24 Yes No A541R NANA NA NA −1.71 0.09 Yes No A541D 0.11 0.12 0.35 0.11 −0.22 0.06 Yes NoA541C NA NA NA NA 0.02 0.39 Yes No A541G 0.38 0.07 0.36 0.07 −0.06 0.12Yes No A541L NA NA NA NA 0.32 0.23 Yes No A541S −0.11 0.15 −0.14 0.15−1.66 0.14 No No A541T −0.11 0.05 −0.11 0.05 −0.50 0.12 No No A541V−0.06 0.09 0.00 0.09 −0.77 0.12 No No S542N 0.24 0.09 0.02 0.09 0.290.07 Yes Yes S542C 0.03 0.17 0.15 0.17 0.42 0.16 Yes Yes S542A NA NA NANA 0.79 0.05 Yes No S542R −0.37 0.10 −0.13 0.10 1.34 0.04 Yes No S542DNA NA NA NA −0.70 0.14 Yes No S542Q NA NA NA NA 0.04 0.23 Yes No S542ENA NA NA NA −1.68 0.07 Yes No S542G 0.23 0.07 −0.10 0.08 −0.79 0.06 YesNo S542H NA NA NA NA 0.08 0.35 Yes No S542I NA NA NA NA −0.13 0.32 YesNo S542L NA NA NA NA 0.67 0.10 Yes No S542K NA NA NA NA 1.36 0.09 Yes NoS542M NA NA NA NA 1.10 0.11 Yes No S542P NA NA NA NA −0.85 0.27 Yes NoS542T 0.68 0.17 0.24 0.19 −0.08 0.10 Yes No S542W NA NA NA NA −0.34 0.06Yes No S542V NA NA NA NA 0.38 0.08 Yes No G543A −2.33 0.07 −1.64 0.05−2.12 0.10 No No G543R −1.78 0.05 −1.75 0.05 −1.89 0.07 No No G543D−1.02 0.07 −1.88 0.09 NA NA Yes No G543C −0.32 0.04 −0.29 0.04 −0.070.06 No No G543E −2.56 0.11 −1.80 0.08 −2.09 0.18 No No G543I −1.30 0.14−1.24 0.13 NA NA Yes No G543L −1.74 0.08 −1.17 0.06 −1.85 0.22 No NoG543F −0.91 0.09 −0.77 0.08 −0.93 0.14 No No G543P −1.28 0.08 −1.18 0.08−1.40 0.22 No No G543S −1.21 0.05 −1.22 0.05 −1.63 0.08 No No G543*−1.66 0.13 −1.93 0.14 NA NA Yes No G543W −1.55 0.08 −1.49 0.07 NA NA YesNo G543V −0.80 0.03 −0.81 0.03 −0.80 0.06 No No W544A −1.17 0.05 −1.900.06 −1.67 0.09 No No W544R −1.30 0.03 −1.40 0.03 −1.35 0.04 No No W544N−1.11 0.10 −1.78 0.13 NA NA Yes No W544D −1.61 0.09 −1.58 0.09 −1.510.14 No No W544C −0.88 0.06 −1.34 0.07 −1.21 0.10 No No W544Q −1.24 0.08−1.17 0.08 NA NA Yes No W544E −1.14 0.05 −1.44 0.05 −1.54 0.06 No NoW544G −1.68 0.03 −1.47 0.03 −1.55 0.04 No No W544H −0.01 0.08 −1.46 0.13NA NA Yes No W544L −1.76 0.06 −1.40 0.05 −1.41 0.04 No No W544K −2.260.11 −1.33 0.07 −1.39 0.08 No No W544M −2.09 0.11 −1.42 0.08 −1.45 0.15No No W544F −0.96 0.09 −1.72 0.12 NA NA Yes No W544P −1.84 0.09 −1.340.08 −1.51 0.09 No No W544S −0.66 0.03 −0.59 0.03 −0.57 0.03 No No W544*−0.85 0.05 −1.05 0.05 −1.09 0.07 No No W544T −1.81 0.09 −1.08 0.07 −1.830.17 No No W544V −1.60 0.05 −1.52 0.05 −1.52 0.09 No No D545A −1.34 0.06−1.18 0.06 −1.75 0.13 No No D545R −2.22 0.08 −1.82 0.06 −1.53 0.12 No NoD545N −1.00 0.10 −0.89 0.09 −1.05 0.15 No No D545C −2.14 0.13 −1.41 0.10NA NA Yes No D545E −1.54 0.07 −2.07 0.09 −1.66 0.14 No No D545G −1.300.04 −1.13 0.04 −1.44 0.07 No No D545H −1.20 0.14 −0.72 0.12 NA NA YesNo D545L −1.77 0.08 −1.23 0.06 −1.75 0.21 No No D545K −1.83 0.15 −1.540.14 NA NA Yes No D545M −1.30 0.11 −0.90 0.10 NA NA Yes No D545P −1.730.12 −1.07 0.09 NA NA Yes No D545S −0.99 0.06 −0.97 0.06 −1.65 0.18 NoNo D545T −2.02 0.14 −1.36 0.10 −1.24 0.25 No No D545W −2.52 0.12 −2.440.12 −1.79 0.16 No No D545Y −1.34 0.12 −1.40 0.12 NA NA Yes No D545V−1.90 0.07 −1.31 0.05 −1.58 0.10 No No V546A 0.05 0.04 −0.28 0.04 −1.030.08 Yes No V546R −0.34 0.04 −0.10 0.04 −0.24 0.05 No No V546D −1.890.14 −0.98 0.10 −1.80 0.23 No No V546C −0.63 0.08 0.12 0.06 −1.17 0.18Yes No V546Q −0.14 0.09 −0.04 0.08 −0.37 0.22 No No V546E −0.85 0.06−0.72 0.06 −1.16 0.10 No No V546G −0.89 0.03 −0.75 0.03 −1.38 0.06 No NoV546I 0.17 0.09 −0.21 0.10 −0.01 0.36 Yes No V546L −0.09 0.04 −0.03 0.04−0.77 0.12 No No V546K −0.23 0.09 −0.07 0.09 −0.28 0.09 No No V546M−0.48 0.06 −0.39 0.06 −0.14 0.11 No No V546F −0.58 0.10 −1.20 0.13 NA NAYes No V546S −0.24 0.05 −0.75 0.06 −1.19 0.07 No No V546* −1.55 0.12−1.19 0.10 −1.56 0.23 No No V546T 0.15 0.07 −0.59 0.09 −0.82 0.09 Yes NoV546W −1.75 0.08 −1.06 0.06 −1.43 0.15 No No V546Y 0.40 0.10 −0.50 0.13−0.45 0.39 Yes No N547A −0.97 0.06 −0.81 0.06 −1.74 0.12 No No N547R−0.75 0.04 −0.98 0.05 −1.25 0.05 No No N547D −1.06 0.08 −0.98 0.08 −1.080.12 No No N547E −1.13 0.07 −1.64 0.09 NA NA Yes No N547G −0.99 0.04−1.38 0.05 −1.68 0.04 No No N547I −0.57 0.10 −0.69 0.10 −0.97 0.15 No NoN547L −2.14 0.12 −1.01 0.07 −1.56 0.18 No No N547K −0.72 0.08 −0.48 0.07−0.83 0.12 No No N547S −0.69 0.06 −0.98 0.06 −1.07 0.09 No No N547T−2.13 0.13 −1.62 0.11 NA NA Yes No N547W −1.69 0.09 −1.84 0.10 NA NA YesNo N547Y −0.39 0.10 −0.94 0.12 −1.04 0.21 No No N547V −1.63 0.07 −1.460.07 −1.86 0.15 No No K548A −1.24 0.04 −1.12 0.04 −1.74 0.03 No No K548R−0.59 0.02 −0.51 0.02 −0.18 0.03 No No K548N −0.98 0.07 −1.11 0.08 −1.100.09 No No K548D −2.69 0.13 −1.63 0.08 −2.01 0.14 No No K548C −0.97 0.06−0.64 0.05 −1.44 0.14 No No K548Q −0.31 0.04 −0.24 0.04 −0.41 0.06 No NoK548E −1.54 0.05 −1.45 0.05 −1.51 0.04 No No K548G −1.14 0.02 −0.95 0.02−1.61 0.02 No No K548H −1.58 0.14 −1.58 0.14 NA NA Yes No K548I −1.050.07 −1.27 0.08 −0.98 0.06 No No K548L −1.72 0.06 −1.56 0.05 −1.96 0.08No No K548M −0.27 0.04 −0.19 0.04 −1.10 0.10 No No K548F −2.01 0.12−1.60 0.10 NA NA Yes No K548P −1.78 0.09 −1.04 0.07 −1.86 0.16 No NoK548S −0.97 0.04 −0.55 0.03 −1.40 0.08 No No K548* −1.65 0.08 −0.85 0.06−1.40 0.13 No No K548T −0.12 0.04 −0.74 0.05 −1.19 0.07 No No K548W−2.13 0.07 −1.65 0.05 −1.44 0.07 No No K548Y −1.07 0.09 −1.63 0.11 NA NAYes No K548V −0.72 0.03 −0.39 0.03 −0.97 0.05 No No E549A −1.80 0.06−1.49 0.05 −1.69 0.07 No No E549R −1.64 0.05 −1.69 0.05 −1.80 0.09 No NoE549D −1.63 0.09 −1.13 0.07 −1.53 0.14 No No E549C −2.08 0.12 −1.84 0.11−1.56 0.18 No No E549Q −0.41 0.05 −0.41 0.05 −0.80 0.10 No No E549G−1.50 0.03 −1.53 0.03 −1.60 0.04 No No E549I −1.07 0.12 −1.12 0.13 NA NAYes No E549L −1.62 0.07 −1.31 0.06 −1.65 0.13 No No E549K −0.91 0.07−0.89 0.07 −0.99 0.08 No No E549M −2.06 0.12 −1.66 0.10 −1.57 0.17 No NoE549F −1.59 0.14 −1.27 0.13 NA NA Yes No E549S −2.21 0.08 −1.69 0.07−1.91 0.05 No No E549* −1.66 0.09 −1.47 0.09 −1.59 0.14 No No E549T NANA NA NA −1.39 0.10 Yes No E549W −0.99 0.05 −1.72 0.07 −1.85 0.11 No NoE549V −1.01 0.04 −0.79 0.04 −1.37 0.04 No No K550R 0.05 0.03 0.18 0.030.17 0.07 Yes Yes K550A −0.24 0.04 −0.06 0.04 −0.91 0.13 No No K550N0.11 0.07 −0.13 0.07 −0.46 0.13 Yes No K550D −0.86 0.09 0.22 0.07 −1.480.23 Yes No K550C −0.08 0.07 −0.18 0.07 −0.84 0.21 No No K550Q −0.180.07 0.45 0.07 −0.64 0.18 Yes No K550E −0.86 0.06 −0.45 0.05 −1.11 0.09No No K550G −0.25 0.02 −0.16 0.02 −1.14 0.04 No No K550H −0.22 0.15−0.42 0.16 NA NA Yes No K550I −0.51 0.12 0.79 0.09 NA NA Yes No K550L0.01 0.05 −0.17 0.05 −0.95 0.06 Yes No K550M −0.71 0.08 −0.17 0.07 −0.720.15 No No K550F −0.14 0.10 −0.39 0.11 NA NA Yes No K550P −0.02 0.08−0.78 0.10 −0.95 0.27 No No K550S −0.07 0.05 −0.22 0.05 −1.05 0.08 No NoK550* −0.98 0.08 −1.14 0.09 −1.19 0.10 No No K550T −0.13 0.06 −0.44 0.07−0.87 0.14 No No K550W 0.18 0.04 0.20 0.04 −0.66 0.07 Yes No K550Y −0.120.11 −0.58 0.12 NA NA Yes No K550V −0.12 0.04 −0.70 0.05 −1.02 0.13 NoNo N551D 0.14 0.04 0.15 0.04 0.67 0.07 Yes Yes N551A −0.29 0.04 −0.420.04 −0.37 0.05 No No N551R −0.92 0.04 −1.40 0.05 −1.64 0.09 No No N551C−0.52 0.08 −0.26 0.08 −0.39 0.28 No No N551Q −0.77 0.09 −0.58 0.08 −0.760.10 No No N551E −1.32 0.06 −0.66 0.05 −1.51 0.10 No No N551G −0.95 0.03−0.96 0.03 −1.10 0.04 No No N551H 0.51 0.09 −1.02 0.14 0.55 0.13 Yes NoN551I −0.01 0.05 0.20 0.05 −0.18 0.11 Yes No N551L −0.07 0.05 −0.83 0.06−1.35 0.11 No No N551K −1.43 0.07 −1.28 0.07 −1.22 0.08 No No N551M−1.01 0.09 −0.58 0.08 −0.94 0.18 No No N551F −0.65 0.11 −0.19 0.09 −0.040.24 No No N551P −1.24 0.10 −0.91 0.09 −1.21 0.12 No No N551S −0.06 0.030.02 0.03 −0.23 0.05 Yes No N551* −1.09 0.10 −1.44 0.11 −1.45 0.20 No NoN551T 0.21 0.05 −0.29 0.06 0.07 0.11 Yes No N551W −0.36 0.06 −0.69 0.06−0.63 0.13 No No N551Y −0.28 0.07 0.04 0.06 0.95 0.10 Yes No N551V −0.310.04 −0.21 0.04 0.03 0.05 Yes No N552A −1.33 0.07 −1.21 0.07 −1.32 0.08No No N552R −1.19 0.04 −1.37 0.05 −1.47 0.04 No No N552D −1.18 0.07−1.04 0.06 −1.24 0.06 No No N552C −1.59 0.10 −1.44 0.09 −1.65 0.17 No NoN552Q −1.21 0.11 −0.81 0.10 NA NA Yes No N552E −1.93 0.09 −0.57 0.05−1.89 0.06 No No N552G −1.80 0.05 −1.17 0.04 −2.06 0.03 No No N552H−0.25 0.10 −0.21 0.10 NA NA Yes No N552I −0.34 0.05 −0.02 0.05 −0.500.09 No No N552L −1.13 0.07 −1.91 0.10 −1.73 0.12 No No N552K −1.34 0.08−1.25 0.08 −1.07 0.10 No No N552M −1.92 0.12 −1.44 0.10 −1.67 0.11 No NoN552P −1.69 0.12 −0.81 0.09 NA NA Yes No N552S −0.11 0.03 −0.20 0.03−0.58 0.05 No No N552* NA NA NA NA −1.37 0.20 Yes No N552T −0.16 0.030.00 0.03 −1.04 0.10 No No N552W −1.97 0.10 −1.69 0.08 −1.53 0.15 No NoN552Y −0.89 0.10 −0.99 0.10 −1.08 0.18 No No N552V −1.60 0.07 −1.17 0.06−1.56 0.05 No No G553A −0.56 0.05 −0.61 0.05 −1.53 0.11 No No G553R−1.58 0.06 −1.17 0.05 −0.39 0.07 No No G553D −0.48 0.08 −0.91 0.09 −0.940.19 No No G553Q −1.38 0.15 −1.10 0.14 NA NA Yes No G553E −2.07 0.11−1.79 0.10 −1.67 0.17 No No G553S −0.78 0.06 −0.54 0.06 −0.72 0.09 No NoG553* −1.04 0.12 −1.39 0.14 NA NA Yes No G553W −1.11 0.07 −1.87 0.09−1.59 0.16 No No G553V −0.34 0.03 −0.24 0.03 −0.30 0.05 No No A554I 0.150.09 0.71 0.08 0.39 0.34 Yes Yes A554T 0.45 0.04 0.32 0.05 0.93 0.07 YesYes A554V 0.57 0.03 0.48 0.03 0.54 0.04 Yes Yes A554R −1.28 0.05 −1.120.05 −2.05 0.11 No No A554N 0.04 0.09 −0.71 0.11 −0.12 0.13 Yes No A554D−1.96 0.10 −0.95 0.07 −0.83 0.04 No No A554C −0.05 0.07 0.32 0.06 1.050.09 Yes No A554Q −0.47 0.10 −0.57 0.10 −1.06 0.28 No No A554E −1.850.09 −0.79 0.06 −1.86 0.11 No No A554G −0.43 0.03 −0.54 0.03 −0.70 0.04No No A554L −0.23 0.05 −0.11 0.05 −0.32 0.17 No No A554M 0.27 0.07 −0.260.08 −0.72 0.23 Yes No A554P −1.28 0.09 −1.84 0.12 NA NA Yes No A554S0.14 0.03 0.15 0.03 −0.07 0.05 Yes No A554* −1.77 0.14 −1.03 0.11 NA NAYes No A554W −1.54 0.09 −2.63 0.14 NA NA Yes No I555V 0.41 0.03 0.130.03 0.14 0.06 Yes Yes I555A −0.39 0.05 −0.48 0.05 −0.79 0.14 No NoI555R −0.47 0.04 −1.21 0.05 −0.58 0.09 No No I555N −0.51 0.08 −0.76 0.08−0.76 0.13 No No I555D −1.00 0.09 −1.17 0.10 NA NA Yes No I555C −1.000.12 −0.93 0.12 −0.75 0.10 No No I555Q −0.79 0.11 −0.92 0.11 −0.84 0.21No No I555E −2.31 0.12 −0.09 0.05 −1.79 0.15 No No I555G −1.15 0.04−1.07 0.04 −1.57 0.03 No No I555L 0.18 0.05 −0.03 0.05 −0.30 0.14 Yes NoI555K −0.71 0.09 −0.03 0.07 −1.33 0.11 No No I555M −0.04 0.05 −0.01 0.05−0.27 0.08 No No I555F −0.18 0.09 −0.17 0.09 −0.32 0.13 No No I555P−1.29 0.11 −1.66 0.13 −1.11 0.36 No No I555S −0.16 0.03 −0.12 0.03 −0.310.05 No No I555T −0.01 0.05 −0.09 0.05 −0.45 0.11 No No I555W −1.78 0.11−1.29 0.09 −1.81 0.15 No No I555Y −1.05 0.14 −1.16 0.15 NA NA Yes NoL556M 0.08 0.08 0.10 0.08 0.11 0.21 Yes Yes L556A −0.52 0.07 −1.77 0.12−1.33 0.24 No No L556R −1.06 0.06 −0.91 0.06 −1.39 0.10 No No L556C−1.31 0.13 −0.58 0.10 NA NA Yes No L556Q −0.06 0.06 −0.34 0.07 −0.360.11 No No L556G −1.55 0.07 −1.49 0.07 −1.60 0.06 No No L556I 0.58 0.120.07 0.14 NA NA Yes No L556P −0.19 0.07 −0.54 0.08 −0.91 0.13 No NoL556S −1.56 0.11 −1.28 0.10 NA NA Yes No L556T −0.95 0.12 −0.69 0.11 NANA Yes No L556W −1.77 0.12 −1.41 0.11 NA NA Yes No L556V −0.45 0.06−0.17 0.05 −0.76 0.14 No No F557A −0.39 0.05 −0.06 0.05 −0.75 0.07 No NoF557R −1.79 0.06 −1.05 0.05 −1.57 0.08 No No F557N NA NA NA NA −0.900.11 Yes No F557C −0.63 0.06 −0.55 0.06 −0.32 0.13 No No F557Q −1.870.15 −0.40 0.09 −0.98 0.25 No No F557E −1.90 0.10 −1.64 0.09 −1.50 0.16No No F557G −1.17 0.04 −1.02 0.04 −0.98 0.06 No No F557I −0.32 0.05−0.07 0.04 −0.05 0.08 No No F557L −0.04 0.04 −0.08 0.04 0.14 0.08 Yes NoF557K −1.25 0.10 −0.84 0.09 −0.79 0.30 No No F557M 0.33 0.07 −0.19 0.08−0.15 0.18 Yes No F557P −1.14 0.10 −1.38 0.11 −1.69 0.21 No No F557S−0.87 0.05 −0.74 0.05 −0.48 0.09 No No F557* −1.30 0.10 −2.32 0.15 NA NAYes No F557T −1.21 0.09 −0.72 0.08 −0.44 0.25 No No F557W −0.87 0.06−0.37 0.05 −1.24 0.11 No No F557Y −0.74 0.08 −0.15 0.07 −0.87 0.13 No NoF557V −0.50 0.04 −0.91 0.05 −0.42 0.04 No No V558M 0.24 0.06 0.15 0.060.10 0.12 Yes Yes V558A −0.11 0.04 −0.13 0.04 0.26 0.09 Yes No V558R−0.63 0.06 −0.46 0.05 −0.26 0.14 No No V558D 0.64 0.09 0.05 0.10 −0.460.14 Yes No V558C −0.01 0.12 0.01 0.12 −0.28 0.55 Yes No V558Q 0.40 0.11−0.65 0.14 −0.21 0.55 Yes No V558E 0.15 0.04 −0.31 0.05 −0.12 0.07 YesNo V558G −0.07 0.03 −0.48 0.04 −0.36 0.10 No No V558L 0.16 0.04 0.030.04 −0.16 0.09 Yes No V558K 0.05 0.10 −0.15 0.11 −0.02 0.48 Yes NoV558F −0.21 0.13 −0.36 0.13 NA NA Yes No V558S −0.36 0.09 −0.49 0.09−0.10 0.27 No No V558T −0.19 0.12 −0.28 0.12 0.15 0.41 Yes No V558W−0.76 0.10 −0.75 0.10 −0.93 0.30 No No K559A 0.17 0.04 0.08 0.04 0.130.08 Yes Yes K559R 0.14 0.03 −0.02 0.03 0.11 0.06 Yes No K559N 0.05 0.030.05 0.03 −0.04 0.05 Yes No K559D 0.25 0.08 −0.33 0.09 −0.39 0.25 Yes NoK559C 0.31 0.08 −0.20 0.09 −0.05 0.22 Yes No K559Q −0.23 0.07 0.03 0.070.00 0.21 Yes No K559E −0.20 0.04 −0.52 0.04 −0.40 0.06 No No K559G−0.35 0.03 −0.41 0.03 −0.16 0.03 No No K559H 0.53 0.14 −0.21 0.16 NA NAYes No K559I −0.62 0.12 0.01 0.10 NA NA Yes No K559L −0.39 0.06 −0.290.06 −0.71 0.13 No No K559M −0.27 0.06 −0.65 0.07 0.01 0.12 Yes No K559F−0.45 0.13 0.13 0.11 NA NA Yes No K559S −0.28 0.06 −0.32 0.06 0.22 0.20Yes No K559* −0.63 0.07 −0.48 0.07 −1.15 0.09 No No K559T 0.30 0.06 0.520.06 −0.20 0.15 Yes No K559W −0.64 0.06 −0.34 0.06 −0.32 0.18 No NoK559Y −0.13 0.11 −0.01 0.11 NA NA Yes No K559V −0.53 0.05 −0.50 0.05−0.27 0.13 No No N560A 0.34 0.05 0.18 0.05 0.18 0.08 Yes Yes N560D 0.160.04 0.18 0.04 0.16 0.08 Yes Yes N560E 0.48 0.06 0.28 0.06 0.11 0.25 YesYes N560M 0.16 0.09 0.31 0.09 0.18 0.13 Yes Yes N560R 0.10 0.04 0.040.04 −0.07 0.06 Yes No N560C −0.11 0.08 −0.20 0.08 −0.15 0.09 No NoN560Q 0.55 0.09 −1.07 0.14 −0.09 0.38 Yes No N560G 0.01 0.04 0.03 0.04−0.02 0.09 Yes No N560H −0.35 0.13 −0.17 0.13 NA NA Yes No N560I 0.220.05 0.28 0.05 −0.13 0.10 Yes No N560L −0.64 0.08 −0.16 0.07 −0.12 0.11No No N560K 0.11 0.06 −0.29 0.07 0.14 0.14 Yes No N560F 0.14 0.12 −0.030.13 NA NA Yes No N560P 0.19 0.09 −0.69 0.11 −0.13 0.29 Yes No N560S0.03 0.03 0.18 0.03 −0.01 0.05 Yes No N560* −1.15 0.11 −1.78 0.15 NA NAYes No N560T −0.40 0.08 0.00 0.07 0.01 0.14 Yes No N560W −0.36 0.07−0.72 0.08 −0.27 0.16 No No N560Y −0.24 0.09 0.04 0.08 0.07 0.12 Yes NoN560V 0.13 0.06 −0.15 0.06 −0.11 0.16 Yes No G561A −0.10 0.05 −0.36 0.06−0.05 0.07 No No G561R −0.20 0.04 0.00 0.04 −0.16 0.05 No No G561N 0.200.16 1.09 0.14 NA NA Yes No G561D −0.43 0.11 0.54 0.08 −0.03 0.27 Yes NoG561C −0.19 0.08 0.03 0.08 −0.26 0.27 Yes No G561Q −0.15 0.12 0.67 0.10−0.08 0.26 Yes No G561E 0.14 0.06 −0.19 0.06 −0.05 0.14 Yes No G561H0.74 0.14 1.07 0.14 NA NA Yes No G561L −0.57 0.09 −0.30 0.09 −0.60 0.09No No G561K 0.24 0.12 0.17 0.12 NA NA Yes No G561M −0.33 0.14 0.12 0.12NA NA Yes No G561P −0.84 0.12 −0.45 0.11 0.02 0.17 Yes No G561S 0.000.06 0.54 0.05 −0.09 0.18 Yes No G561* −0.53 0.11 −0.01 0.09 NA NA YesNo G561T 0.10 0.09 −0.87 0.13 −0.23 0.34 Yes No G561W −0.47 0.06 −0.340.06 −0.51 0.17 No No G561V −0.02 0.02 −0.04 0.02 0.08 0.06 Yes No L562A−0.46 0.07 −0.26 0.07 −0.17 0.22 No No L562R −0.38 0.05 0.08 0.04 −0.170.08 Yes No L562D −0.37 0.13 −0.90 0.15 −0.46 0.35 No No L562C 0.18 0.10−0.63 0.12 0.13 0.29 Yes No L562Q −0.43 0.09 −0.20 0.08 −0.07 0.17 No NoL562E −0.32 0.07 −0.62 0.08 −0.29 0.18 No No L562G −0.75 0.05 −0.60 0.05−0.36 0.08 No No L562H 0.23 0.12 −0.50 0.15 NA NA Yes No L562K −0.690.12 0.32 0.09 −0.53 0.29 Yes No L562M 0.19 0.06 −0.26 0.07 −0.04 0.12Yes No L562F 0.48 0.14 0.79 0.13 NA NA Yes No L562P −0.10 0.06 −0.050.06 −0.26 0.09 No No L562S −0.65 0.10 −0.27 0.09 −0.04 0.26 No No L562*−1.29 0.14 −1.10 0.13 NA NA Yes No L562T −1.14 0.15 −1.09 0.15 NA NA YesNo L562W −0.37 0.07 −0.38 0.07 −0.45 0.18 No No L562Y −0.63 0.16 −0.490.16 NA NA Yes No L562V −0.20 0.05 −0.49 0.06 −0.71 0.11 No No Y563A−1.58 0.09 −1.18 0.08 −1.03 0.07 No No Y563R −1.46 0.06 −1.08 0.05 −1.590.11 No No Y563N −0.16 0.08 0.04 0.07 −0.30 0.16 Yes No Y563D −0.56 0.07−0.65 0.07 −0.73 0.07 No No Y563C −0.49 0.06 −0.50 0.06 −0.32 0.09 No NoY563Q −0.48 0.10 −0.52 0.10 −0.93 0.25 No No Y563E −1.21 0.08 0.07 0.06−1.73 0.14 Yes No Y563G −1.13 0.04 −1.64 0.05 −1.26 0.03 No No Y563H−0.25 0.08 −0.14 0.08 −0.06 0.15 No No Y563L −1.95 0.12 −1.69 0.11 −1.150.19 No No Y563F 0.13 0.07 0.06 0.07 −0.06 0.11 Yes No Y563S −0.91 0.07−0.95 0.07 −0.96 0.08 No No Y563* −0.54 0.08 −0.93 0.09 −1.33 0.14 No NoY563T −1.67 0.15 0.09 0.08 −0.51 0.22 Yes No Y563W −0.84 0.06 −0.78 0.05−1.12 0.11 No No Y563V −1.32 0.08 −1.12 0.07 −1.13 0.17 No No Y564A−1.20 0.15 −0.93 0.14 NA NA Yes No Y564R −1.37 0.10 −1.99 0.13 −1.190.19 No No Y564N −0.18 0.12 0.08 0.11 −0.14 0.21 Yes No Y564D −0.05 0.06−0.60 0.07 −0.25 0.10 No No Y564C 0.09 0.05 −0.22 0.05 −0.08 0.10 Yes NoY564G −1.96 0.11 −1.74 0.10 −1.32 0.19 No No Y564H −0.05 0.08 −0.10 0.080.04 0.13 Yes No Y564L −0.24 0.10 −1.01 0.13 NA NA Yes No Y564F −0.040.11 0.35 0.10 NA NA Yes No Y564S −0.47 0.09 −0.36 0.08 −0.35 0.20 No NoY564* −0.43 0.11 −0.68 0.12 NA NA Yes No Y564W −0.37 0.10 0.03 0.09−0.52 0.29 Yes No Y564V −1.90 0.15 −0.72 0.10 NA NA Yes No L565A −0.650.07 −0.47 0.06 −1.27 0.23 No No L565R −0.27 0.03 −0.79 0.03 −0.12 0.05No No L565C −1.21 0.13 −0.10 0.09 −0.66 0.24 No No L565Q −0.36 0.08−0.84 0.09 −0.76 0.15 No No L565E −1.93 0.12 −1.02 0.09 −1.55 0.19 No NoL565G −2.36 0.08 −1.62 0.06 −1.50 0.10 No No L565I −0.07 0.13 −0.42 0.14NA NA Yes No L565M −0.20 0.07 −0.26 0.07 0.03 0.10 Yes No L565P −0.600.07 −0.92 0.08 −0.67 0.13 No No L565S −1.07 0.09 −0.75 0.08 −1.13 0.08No No L565W −1.64 0.10 −2.31 0.13 NA NA Yes No L565V −0.57 0.06 −0.480.05 −1.19 0.13 No No G566A −0.36 0.08 −0.39 0.08 −0.57 0.21 No No G566R−1.37 0.09 −1.97 0.12 −1.83 0.11 No No G566D −0.67 0.11 −1.03 0.12 −0.620.18 No No G566C −0.08 0.11 −0.83 0.13 NA NA Yes No G566S −0.14 0.06−0.40 0.07 −0.18 0.11 No No G566V −0.52 0.07 −0.99 0.08 −0.68 0.10 No NoI567A −1.35 0.11 −1.01 0.09 −1.13 0.07 No No I567R −1.78 0.09 −0.11 0.05−1.44 0.11 No No I567N −0.05 0.07 −0.10 0.07 0.15 0.10 Yes No I567E−1.64 0.12 −1.70 0.13 NA NA Yes No I567G −1.23 0.06 −1.14 0.06 −1.700.09 No No I567L −0.67 0.08 0.29 0.06 −0.48 0.13 Yes No I567M −0.51 0.09−0.82 0.10 −0.88 0.10 No No I567F −0.43 0.12 0.00 0.11 −0.48 0.19 No NoI567S −0.56 0.07 −0.67 0.07 −0.82 0.12 No No I567T −0.15 0.05 −0.27 0.060.13 0.09 Yes No I567W −1.85 0.12 −2.17 0.14 −1.22 0.13 No No I567V−0.54 0.05 −0.26 0.05 −0.68 0.08 No No M568A −0.52 0.06 −0.34 0.06 −0.040.11 No No M568R −0.30 0.04 −0.82 0.05 −0.20 0.07 No No M568C −0.33 0.10−0.78 0.11 −0.18 0.28 No No M568Q −0.61 0.12 −0.40 0.12 NA NA Yes NoM568E −1.29 0.09 −0.86 0.08 −1.65 0.22 No No M568G −1.32 0.05 −0.45 0.04−0.80 0.04 No No M568H 0.40 0.14 −0.11 0.15 NA NA Yes No M568I 0.16 0.05−0.01 0.06 0.15 0.11 Yes No M568L −0.42 0.06 −0.23 0.05 −0.27 0.09 No NoM568K −0.19 0.08 −0.20 0.08 −0.17 0.16 No No M568P −1.11 0.13 −0.81 0.12NA NA Yes No M568S 0.29 0.06 −0.47 0.07 −0.54 0.22 Yes No M568T −0.130.06 −0.25 0.06 −0.07 0.12 No No M568W −0.20 0.06 0.14 0.05 −0.93 0.18Yes No M568V −0.26 0.04 −0.28 0.04 0.06 0.08 Yes No P569D 0.60 0.08 0.330.08 1.25 0.11 Yes Yes P569A −0.12 0.05 −0.39 0.05 0.23 0.08 Yes NoP569R −0.60 0.04 −0.52 0.04 −0.13 0.05 No No P569N −0.14 0.14 −0.71 0.16NA NA Yes No P569C −1.16 0.09 −0.36 0.07 0.07 0.19 Yes No P569Q −0.260.09 −0.13 0.09 0.40 0.21 Yes No P569E −0.07 0.07 −0.16 0.07 −0.11 0.22No No P569G −0.54 0.03 −0.49 0.03 −0.65 0.07 No No P569H 0.12 0.09 −0.250.10 0.16 0.17 Yes No P569I 0.03 0.13 −0.32 0.14 0.10 0.31 Yes No P569L−0.47 0.05 −0.21 0.04 −0.30 0.11 No No P569K −0.35 0.12 0.36 0.10 0.230.31 Yes No P569M −0.98 0.11 −0.54 0.10 −0.28 0.12 No No P569F NA NA NANA −0.67 0.31 Yes No P569S −0.07 0.05 −0.24 0.05 0.02 0.11 Yes No P569T−0.38 0.07 −0.20 0.07 −0.06 0.15 No No P569W −0.74 0.06 −1.07 0.07 −1.180.14 No No P569V −0.34 0.04 −0.82 0.05 0.15 0.14 Yes No K570A −0.95 0.06−0.28 0.05 −1.70 0.16 No No K570R −0.47 0.03 −0.56 0.04 −0.39 0.05 No NoK570N −0.30 0.11 −0.73 0.12 NA NA Yes No K570D −1.38 0.12 −1.42 0.13 NANA Yes No K570C −1.71 0.11 −1.70 0.11 NA NA Yes No K570Q −0.90 0.09−0.32 0.08 −1.25 0.18 No No K570E −1.37 0.07 −0.71 0.05 −1.40 0.10 No NoK570G −0.62 0.03 −0.33 0.03 −1.61 0.09 No No K570L −1.83 0.08 −1.48 0.07−1.35 0.14 No No K570M −1.02 0.09 −0.76 0.09 −1.52 0.18 No No K570P−1.15 0.12 −0.12 0.09 NA NA Yes No K570S −1.28 0.09 −0.51 0.07 −1.520.14 No No K570* −1.31 0.10 −1.26 0.10 −1.24 0.15 No No K570T −0.02 0.030.11 0.03 −0.05 0.03 Yes No K570W −2.06 0.09 −2.64 0.12 −1.61 0.14 No NoK570V −1.33 0.06 −0.81 0.05 −1.85 0.11 No No Q571A 0.64 0.07 0.39 0.081.09 0.23 Yes Yes Q571P 0.17 0.07 0.61 0.07 0.13 0.14 Yes Yes Q571S 0.250.09 0.20 0.09 0.89 0.23 Yes Yes Q571T 0.22 0.13 0.19 0.13 0.87 0.41 YesYes Q571R −0.03 0.04 −0.43 0.05 0.19 0.10 Yes No Q571C −0.60 0.16 0.230.12 −0.33 0.24 Yes No Q571E −0.22 0.05 −0.40 0.06 −0.17 0.05 No NoQ571G 0.00 0.06 −0.25 0.06 −0.41 0.18 No No Q571H −0.23 0.11 −0.65 0.120.06 0.24 Yes No Q571L −0.41 0.07 −0.45 0.07 −0.66 0.11 No No Q571K−0.47 0.16 −0.33 0.15 0.08 0.31 Yes No Q571* −0.59 0.11 −0.27 0.10 −0.670.17 No No Q571W −0.09 0.08 −0.80 0.09 −0.51 0.24 No No Q571V −0.36 0.11−0.20 0.10 −0.44 0.32 No No K572E 0.11 0.04 0.25 0.04 0.42 0.08 Yes YesK572A −0.24 0.04 0.33 0.04 −0.11 0.11 Yes No K572R −0.08 0.03 −0.05 0.030.03 0.03 Yes No K572N −0.17 0.07 0.17 0.06 0.02 0.11 Yes No K572D −0.090.09 0.03 0.08 0.45 0.19 Yes No K572C 0.23 0.07 −0.87 0.09 −0.19 0.25Yes No K572Q −0.21 0.06 0.11 0.06 0.37 0.16 Yes No K572G −0.17 0.03−0.08 0.03 0.42 0.03 Yes No K572H NA NA NA NA 0.11 0.33 Yes No K572L−0.11 0.05 0.16 0.05 −0.32 0.15 Yes No K572M −0.71 0.08 0.02 0.06 0.210.06 Yes No K572P 0.09 0.06 0.91 0.06 −0.88 0.23 Yes No K572S 0.30 0.050.01 0.05 −0.29 0.15 Yes No K572* −1.34 0.08 −0.86 0.07 −0.86 0.08 No NoK572T −0.04 0.04 −0.09 0.04 −0.24 0.07 No No K572W −0.46 0.05 −0.47 0.05−0.68 0.14 No No K572Y −1.04 0.16 −1.01 0.15 NA NA Yes No K572V −0.080.04 −0.31 0.04 −0.71 0.04 No No G573A 0.04 0.04 −0.11 0.04 −0.99 0.09Yes No G573R −0.04 0.03 −0.31 0.04 −0.66 0.08 No No G573N 0.45 0.12−0.94 0.17 −0.29 0.41 Yes No G573D −0.05 0.06 −0.41 0.07 −0.95 0.13 NoNo G573C −0.22 0.06 −0.35 0.06 −0.42 0.11 No No G573Q 0.40 0.07 −0.290.08 −0.43 0.26 Yes No G573E −0.09 0.05 0.36 0.05 −0.50 0.08 Yes NoG573H −0.77 0.12 0.37 0.09 −0.51 0.30 Yes No G573L −0.04 0.05 0.07 0.05−0.43 0.05 Yes No G573K −1.43 0.13 −0.29 0.09 −0.32 0.25 No No G573M−0.32 0.10 −0.32 0.09 −0.28 0.26 No No G573F −0.45 0.14 −0.19 0.12 NA NAYes No G573P 0.16 0.06 −0.38 0.06 −0.10 0.21 Yes No G573S 0.11 0.04−0.42 0.05 −0.55 0.10 Yes No G573* −1.18 0.12 −1.27 0.12 −0.75 0.28 NoNo G573T 0.02 0.08 −0.53 0.09 −0.62 0.08 Yes No G573W −0.08 0.05 −0.600.05 −0.67 0.09 No No G573Y 0.13 0.11 −0.81 0.14 −0.48 0.30 Yes No G573V−0.06 0.04 −0.40 0.04 −0.71 0.06 No No R574A −0.83 0.07 −0.54 0.06 −0.640.10 No No R574N −0.85 0.17 −0.69 0.16 NA NA Yes No R574D −0.32 0.10−1.05 0.13 NA NA Yes No R574C 0.58 0.07 0.15 0.07 −1.16 0.22 Yes NoR574Q NA NA NA NA −1.08 0.26 Yes No R574E −0.53 0.08 −0.38 0.07 −1.340.15 No No R574G −0.21 0.03 −0.27 0.03 −0.60 0.07 No No R574I −0.50 0.11−0.47 0.11 NA NA Yes No R574L −0.36 0.07 −0.25 0.07 −0.78 0.18 No NoR574K −0.33 0.08 0.42 0.06 −0.33 0.17 Yes No R574M 0.51 0.07 −0.36 0.08−0.64 0.07 Yes No R574F 0.06 0.12 −0.99 0.16 NA NA Yes No R574P −0.380.13 0.11 0.11 −0.72 0.23 Yes No R574S 0.12 0.04 0.02 0.04 −1.04 0.03Yes No R574* −0.34 0.07 −0.51 0.07 −0.84 0.09 No No R574T −0.65 0.090.35 0.07 −0.83 0.16 Yes No R574W −0.10 0.05 −0.10 0.05 −0.73 0.06 No NoR574Y −0.25 0.12 0.13 0.11 NA NA Yes No R574V 0.59 0.05 −0.27 0.05 −1.080.14 Yes No Y575G 0.53 0.04 0.21 0.04 0.32 0.07 Yes Yes Y575M 0.89 0.110.27 0.12 0.31 0.41 Yes Yes Y575A 0.55 0.05 0.01 0.06 −0.07 0.18 Yes NoY575R 0.15 0.05 −0.04 0.06 −0.56 0.10 Yes No Y575N −0.07 0.10 0.34 0.09−0.21 0.10 Yes No Y575D −0.07 0.09 0.15 0.09 −0.45 0.25 Yes No Y575C0.20 0.06 0.17 0.06 −0.26 0.09 Yes No Y575Q −0.22 0.13 −0.32 0.13 −0.160.30 No No Y575E 0.03 0.08 −0.32 0.09 −0.41 0.35 Yes No Y575H −0.24 0.08−0.29 0.08 0.23 0.13 Yes No Y575I 0.04 0.14 −0.37 0.15 NA NA Yes NoY575L −0.05 0.07 −0.43 0.08 −0.22 0.23 No No Y575K 0.95 0.11 0.22 0.12NA NA Yes No Y575F 0.14 0.08 −0.45 0.09 −0.16 0.12 Yes No Y575P −0.060.09 −0.22 0.09 −0.14 0.31 No No Y575S 0.02 0.06 0.01 0.06 −0.07 0.09Yes No Y575* −0.20 0.07 −0.46 0.08 −0.51 0.12 No No Y575T −0.55 0.10−0.40 0.09 −0.14 0.30 No No Y575W −0.44 0.09 0.31 0.07 −0.36 0.16 Yes NoY575V −0.10 0.07 −0.35 0.07 −0.29 0.24 No No K576C 0.27 0.07 0.25 0.070.10 0.26 Yes Yes K576A −0.05 0.05 0.16 0.05 0.27 0.06 Yes No K576R−0.31 0.03 −0.11 0.03 0.05 0.07 Yes No K576N −0.28 0.08 −0.47 0.08 −0.110.15 No No K576D −0.01 0.08 −0.22 0.09 −1.04 0.26 No No K576Q −0.06 0.060.02 0.05 0.57 0.08 Yes No K576E −0.63 0.05 −0.38 0.05 −0.27 0.09 No NoK576G −0.03 0.03 −0.13 0.03 0.28 0.04 Yes No K576H −0.26 0.14 −0.46 0.14NA NA Yes No K576I 0.10 0.11 −0.10 0.12 0.37 0.16 Yes No K576L −0.040.05 0.01 0.04 0.24 0.05 Yes No K576M 0.21 0.05 −0.14 0.06 0.07 0.09 YesNo K576F −0.41 0.13 −1.12 0.15 0.20 0.34 Yes No K576P −0.07 0.09 −1.700.14 −0.15 0.17 No No K576S −0.20 0.05 −0.43 0.06 0.64 0.15 Yes No K576*−1.75 0.10 −1.41 0.08 −0.09 0.15 No No K576T −0.05 0.06 0.30 0.05 0.200.08 Yes No K576W −0.33 0.05 −0.65 0.05 −0.56 0.13 No No K576Y −1.230.16 −0.48 0.12 NA NA Yes No K576V −0.36 0.04 −0.69 0.05 0.38 0.07 YesNo A577R −0.12 0.03 −0.60 0.04 0.88 0.06 Yes No A577N 0.11 0.09 −0.850.11 0.34 0.13 Yes No A577D −0.06 0.05 −0.11 0.05 −0.37 0.13 No No A577C−0.24 0.06 −0.02 0.06 −0.39 0.20 No No A577Q −0.37 0.07 0.63 0.06 0.100.23 Yes No A577E −0.66 0.05 −2.12 0.09 −0.27 0.14 No No A577G 0.22 0.02−0.05 0.03 0.49 0.05 Yes No A577H 0.02 0.08 −0.59 0.10 0.25 0.22 Yes NoA577I −0.94 0.12 −0.35 0.10 NA NA Yes No A577L −0.90 0.06 −0.37 0.05−0.15 0.06 No No A577K −0.40 0.08 −0.80 0.09 1.09 0.07 Yes No A577M−0.40 0.08 −0.20 0.07 −0.28 0.20 No No A577F −0.79 0.11 −0.99 0.12 −0.780.19 No No A577P −0.25 0.06 −0.11 0.05 −0.29 0.11 No No A577S −0.31 0.04−0.26 0.04 0.08 0.10 Yes No A577* −1.65 0.11 −1.75 0.11 NA NA Yes NoA577T −0.09 0.05 −0.38 0.05 −0.02 0.08 No No A577W −0.63 0.05 −0.66 0.05−0.66 0.08 No No A577Y −0.22 0.09 −0.20 0.09 0.05 0.32 Yes No A577V−0.06 0.03 −0.28 0.04 −0.42 0.09 No No L578A −0.22 0.03 −0.43 0.03 −0.200.07 No No L578R −0.11 0.02 −0.49 0.02 0.04 0.03 Yes No L578N −0.35 0.11−0.09 0.10 −0.47 0.23 No No L578D −0.09 0.06 −0.41 0.06 −0.92 0.07 No NoL578C 0.05 0.04 −0.34 0.05 −0.03 0.10 Yes No L578Q 0.11 0.04 −0.12 0.050.00 0.10 Yes No L578E −0.33 0.04 −0.53 0.04 −0.77 0.12 No No L578G−0.26 0.02 −0.45 0.02 −0.29 0.02 No No L578H −0.27 0.07 0.10 0.06 0.030.14 Yes No L578I −0.28 0.09 0.32 0.08 0.22 0.23 Yes No L578K −0.62 0.07−1.21 0.09 0.50 0.20 Yes No L578M −0.12 0.03 −0.21 0.03 −0.03 0.05 No NoL578F 0.36 0.06 0.21 0.06 −0.12 0.14 Yes No L578P −0.62 0.05 0.03 0.04−0.31 0.06 Yes No L578S −0.37 0.04 −0.33 0.04 −0.08 0.09 No No L578*−1.90 0.09 −1.28 0.07 −1.24 0.09 No No L578T −0.29 0.06 −0.06 0.05 0.260.06 Yes No L578W −0.37 0.03 −0.41 0.03 −0.08 0.04 No No L578Y 0.36 0.07−0.21 0.07 −0.24 0.07 Yes No L578V −0.19 0.03 −0.28 0.03 0.01 0.04 YesNo S579T 0.17 0.04 0.33 0.04 0.01 0.06 Yes Yes S579V 0.04 0.03 0.02 0.030.05 0.07 Yes Yes S579A −0.06 0.03 0.06 0.02 0.08 0.03 Yes No S579R 0.040.03 −0.11 0.03 −0.15 0.05 Yes No S579N −0.19 0.07 −0.45 0.08 −0.39 0.23No No S579D −0.39 0.06 −0.43 0.06 0.03 0.06 Yes No S579C 0.02 0.03 0.070.03 −0.02 0.05 Yes No S579Q −0.19 0.07 −0.66 0.08 0.30 0.20 Yes NoS579E −0.10 0.04 −0.54 0.05 −0.15 0.05 No No S579G −0.25 0.03 −0.12 0.020.11 0.05 Yes No S579H 0.09 0.08 −0.15 0.08 −0.18 0.12 Yes No S579I−0.05 0.06 0.25 0.06 0.04 0.15 Yes No S579L −0.65 0.04 −0.32 0.04 0.110.05 Yes No S579K −0.15 0.06 −0.55 0.07 0.13 0.22 Yes No S579M −0.180.06 0.05 0.05 0.28 0.19 Yes No S579F 0.27 0.04 0.16 0.04 −0.08 0.07 YesNo S579P −0.19 0.05 −0.75 0.05 −0.05 0.10 No No S579* −1.34 0.08 −1.060.07 −1.30 0.11 No No S579W −0.09 0.04 −0.35 0.04 −0.37 0.06 No No S579Y−0.06 0.05 −0.21 0.05 −0.36 0.04 No No F580A −0.80 0.08 −0.25 0.07 −1.440.19 No No F580R −1.58 0.09 −1.48 0.08 −1.26 0.19 No No F580C −0.22 0.09−0.47 0.10 −0.11 0.18 No No F580E −0.96 0.12 −0.21 0.09 NA NA Yes NoF580G −0.91 0.05 −1.05 0.05 −1.21 0.04 No No F580I −0.14 0.09 −0.06 0.09−0.17 0.28 No No F580L −0.11 0.05 −0.28 0.05 0.01 0.09 Yes No F580M 0.310.12 −0.37 0.14 NA NA Yes No F580P NA NA NA NA −0.75 0.28 Yes No F580S−0.22 0.07 −0.70 0.07 −0.67 0.10 No No F580W −1.14 0.10 0.30 0.07 0.670.24 Yes No F580Y 0.39 0.11 −0.45 0.14 −0.09 0.23 Yes No F580V 0.18 0.06−0.33 0.06 −0.52 0.13 Yes No E581A −0.08 0.03 0.16 0.03 −0.13 0.06 YesNo E581R −0.31 0.03 −0.14 0.03 −0.36 0.04 No No E581N −0.14 0.15 0.560.13 NA NA Yes No E581D 0.04 0.06 0.25 0.06 −0.21 0.09 Yes No E581C 0.120.07 0.54 0.06 −0.12 0.15 Yes No E581Q 0.01 0.04 0.09 0.04 −0.12 0.06Yes No E581G −0.15 0.02 −0.05 0.02 −0.03 0.03 No No E581H −0.31 0.121.00 0.10 −0.67 0.35 Yes No E581I −0.12 0.14 0.03 0.13 0.40 0.44 Yes NoE581L 0.09 0.04 −0.12 0.04 0.29 0.12 Yes No E581K −0.10 0.05 −0.02 0.05−0.33 0.07 No No E581M −0.05 0.08 −0.79 0.09 0.12 0.13 Yes No E581F−0.32 0.11 −0.09 0.10 −0.40 0.30 No No E581P −0.49 0.07 −0.09 0.06 −0.250.13 No No E581S 0.00 0.05 −1.01 0.06 −0.11 0.06 Yes No E581* −1.00 0.07−0.93 0.07 −0.92 0.13 No No E581T −0.23 0.07 −0.29 0.07 0.06 0.09 Yes NoE581W 0.25 0.04 −0.31 0.04 0.04 0.05 Yes No E581Y NA NA NA NA 0.25 0.28Yes No E581V −0.18 0.04 0.31 0.03 0.26 0.10 Yes No P582A −0.46 0.07−0.64 0.07 −0.60 0.13 No No P582R 0.24 0.05 −0.24 0.06 −0.48 0.17 Yes NoP582N −0.02 0.05 −0.24 0.05 −0.22 0.11 No No P582D −0.41 0.18 −0.64 0.18NA NA Yes No P582C 0.14 0.11 −0.07 0.12 0.17 0.64 Yes No P582Q −0.740.13 −0.13 0.11 −0.07 0.25 No No P582G −0.10 0.06 −0.80 0.07 −0.50 0.18No No P582H −0.03 0.02 −0.15 0.02 −0.12 0.04 No No P582L −0.14 0.07−0.96 0.08 −0.26 0.13 No No P582K −0.33 0.19 0.97 0.15 NA NA Yes NoP582F NA NA NA NA 0.02 0.55 Yes No P582S 0.19 0.06 0.48 0.05 −1.33 0.16Yes No P582T −0.13 0.03 −0.19 0.03 −0.48 0.06 No No P582W −1.46 0.15−0.73 0.11 −0.12 0.35 No No P582V −0.90 0.10 −0.27 0.08 −0.81 0.09 No NoT583I 0.04 0.08 0.11 0.07 0.07 0.17 Yes Yes T583A 0.02 0.04 −0.22 0.04−0.17 0.08 Yes No T583R 0.12 0.05 −0.31 0.05 −0.04 0.09 Yes No T583N0.00 0.06 0.01 0.06 −0.20 0.09 Yes No T583D 0.23 0.12 −0.96 0.16 NA NAYes No T583C 0.67 0.09 −0.82 0.12 −0.67 0.27 Yes No T583Q NA NA NA NA−0.28 0.36 Yes No T583E −0.62 0.10 0.01 0.09 −0.72 0.28 Yes No T583G−0.17 0.05 −0.35 0.05 0.16 0.06 Yes No T583L −0.09 0.06 −0.72 0.07 −0.240.22 No No T583K NA NA NA NA −0.15 0.44 Yes No T583M −0.72 0.13 −0.890.14 −0.12 0.32 No No T583F −0.41 0.13 −0.83 0.15 −0.13 0.36 No No T583P−0.14 0.08 −0.64 0.08 −0.37 0.14 No No T583S 0.18 0.05 0.11 0.05 −0.340.07 Yes No T583W −0.31 0.07 −0.54 0.08 −0.09 0.08 No No T583V 0.01 0.06−0.05 0.06 0.15 0.22 Yes No E584H 0.09 0.07 0.22 0.07 0.17 0.14 Yes YesE584V 0.10 0.03 0.06 0.03 0.12 0.04 Yes Yes E584A −0.01 0.03 0.09 0.030.10 0.06 Yes No E584R −0.23 0.03 −0.12 0.03 −0.10 0.05 No No E584N−0.50 0.09 −0.38 0.08 −0.11 0.20 No No E584D 0.46 0.04 0.51 0.04 −0.020.08 Yes No E584C −0.03 0.05 0.09 0.05 0.13 0.07 Yes No E584Q 0.09 0.07−0.88 0.08 −0.09 0.10 Yes No E584G −0.02 0.02 0.06 0.02 0.16 0.04 Yes NoE584I 0.32 0.07 −0.34 0.08 −0.03 0.20 Yes No E584L −0.27 0.04 0.00 0.04−0.06 0.10 No No E584K −0.29 0.06 −0.78 0.07 0.39 0.09 Yes No E584M−0.17 0.07 −0.68 0.08 0.34 0.13 Yes No E584F −0.11 0.08 −0.41 0.09 0.270.19 Yes No E584P 0.12 0.05 −0.13 0.06 −0.24 0.17 Yes No E584S −0.440.04 0.09 0.04 0.26 0.09 Yes No E584* −1.22 0.09 −1.26 0.08 −1.15 0.13No No E584T −0.03 0.05 −0.03 0.05 0.17 0.15 Yes No E584W −0.73 0.06−0.52 0.05 −0.32 0.07 No No E584Y −0.13 0.07 0.55 0.06 0.05 0.25 Yes NoK585R 0.07 0.03 0.32 0.03 0.06 0.05 Yes Yes K585F 0.51 0.09 0.04 0.090.61 0.36 Yes Yes K585A 0.21 0.04 −0.23 0.05 0.16 0.13 Yes No K585N−0.13 0.09 0.18 0.08 −0.67 0.12 Yes No K585D −0.05 0.07 −0.08 0.07 −0.050.23 No No K585C −0.25 0.07 −0.61 0.08 −0.11 0.29 No No K585Q 0.07 0.060.41 0.06 −0.09 0.08 Yes No K585E −0.33 0.05 −0.44 0.05 0.10 0.12 Yes NoK585G −0.18 0.03 −0.04 0.03 0.16 0.04 Yes No K585H 0.12 0.11 −0.60 0.130.28 0.14 Yes No K585I −1.45 0.14 0.35 0.08 0.11 0.25 Yes No K585L −0.100.04 −0.06 0.04 0.27 0.13 Yes No K585M −0.25 0.07 −0.05 0.06 0.21 0.17Yes No K585P 0.20 0.06 −0.04 0.07 0.15 0.21 Yes No K585S −0.17 0.05 0.240.04 0.20 0.11 Yes No K585* −0.91 0.07 −0.60 0.06 −0.61 0.11 No No K585T0.08 0.05 −0.30 0.05 0.05 0.07 Yes No K585W −0.21 0.05 −0.15 0.05 0.180.16 Yes No K585Y 0.18 0.10 −0.92 0.13 0.07 0.40 Yes No K585V −0.16 0.04−0.68 0.04 0.37 0.06 Yes No T586A −0.30 0.05 −0.02 0.04 −0.01 0.11 No NoT586R −0.56 0.05 −0.06 0.04 −0.20 0.14 No No T586N −0.07 0.05 0.15 0.05−0.02 0.07 Yes No T586D −0.41 0.12 1.10 0.09 0.41 0.30 Yes No T586C−0.60 0.11 −0.45 0.10 0.10 0.29 Yes No T586Q −0.65 0.14 0.28 0.11 NA NAYes No T586E −0.29 0.09 0.04 0.08 0.16 0.37 Yes No T586G −0.21 0.04 0.220.03 −0.07 0.09 Yes No T586I −0.77 0.11 0.90 0.07 −0.48 0.19 Yes NoT586L −0.06 0.06 −0.85 0.07 0.01 0.18 Yes No T586K −0.06 0.12 0.80 0.10−0.12 0.13 Yes No T586M −0.62 0.11 −1.06 0.13 0.22 0.34 Yes No T586F0.21 0.13 −0.21 0.14 0.02 0.54 Yes No T586P 0.38 0.06 −0.09 0.06 0.220.12 Yes No T586S −0.22 0.05 −0.08 0.05 0.01 0.08 Yes No T586W −0.480.07 −0.61 0.07 −0.29 0.24 No No T586Y NA NA NA NA 0.24 0.26 Yes NoT586V −0.97 0.07 −0.13 0.05 0.13 0.17 Yes No S587A NA NA NA NA 0.45 0.32Yes No S587R 0.15 0.08 −0.61 0.09 −0.34 0.15 Yes No S587N −0.30 0.13−0.31 0.13 −0.26 0.18 No No S587C 0.01 0.15 0.00 0.14 NA NA Yes No S587E0.33 0.16 −0.40 0.19 NA NA Yes No S587G −0.32 0.06 −0.81 0.07 −0.21 0.06No No S587L 0.21 0.14 0.27 0.13 NA NA Yes No S587T 0.80 0.11 −0.41 0.14−0.13 0.23 Yes No S587V 0.20 0.11 −0.25 0.12 −0.04 0.12 Yes No E588A−0.01 0.05 0.25 0.05 0.17 0.10 Yes No E588R −0.39 0.05 −0.61 0.05 −0.310.07 No No E588D 0.00 0.08 −0.12 0.08 −0.55 0.13 Yes No E588C −0.64 0.11−0.35 0.10 0.00 0.10 No No E588Q 0.11 0.09 −0.25 0.09 −0.08 0.13 Yes NoE588G −0.16 0.04 −0.19 0.04 −0.34 0.04 No No E588H −0.35 0.17 −0.26 0.16NA NA Yes No E588L −0.31 0.07 −0.29 0.07 0.41 0.12 Yes No E588K 0.180.07 0.13 0.07 −0.25 0.15 Yes No E588M −0.50 0.12 −0.10 0.11 0.54 0.12Yes No E588F −0.23 0.15 0.13 0.14 NA NA Yes No E588P −0.04 0.10 0.060.09 0.52 0.19 Yes No E588S −0.03 0.07 −0.34 0.08 −0.47 0.08 No No E588*−1.05 0.11 −1.06 0.11 −1.02 0.15 No No E588T 0.32 0.09 −1.53 0.15 NA NAYes No E588W −0.02 0.07 0.03 0.07 −0.01 0.19 Yes No E588Y −0.11 0.170.93 0.14 NA NA Yes No E588V −0.70 0.06 −0.19 0.05 0.07 0.08 Yes NoG589A −0.44 0.05 −0.09 0.04 −0.52 0.14 No No G589R −0.21 0.04 −0.73 0.04−0.69 0.07 No No G589D −0.29 0.08 −0.88 0.10 −0.70 0.17 No No G589C−0.14 0.04 −0.12 0.04 −0.59 0.07 No No G589Q NA NA NA NA −0.13 0.27 YesNo G589E −0.39 0.06 −1.47 0.08 −0.59 0.24 No No G589L 0.07 0.06 −1.020.08 −1.07 0.20 Yes No G589M −0.45 0.12 −1.01 0.14 NA NA Yes No G589P−0.34 0.08 −2.17 0.15 −1.08 0.12 No No G589S −0.13 0.04 −0.30 0.04 −0.540.10 No No G589T −0.52 0.11 −0.51 0.10 −1.12 0.28 No No G589W −0.62 0.06−0.77 0.07 −0.26 0.13 No No G589Y NA NA NA NA −0.01 0.41 Yes No G589V−0.65 0.05 −0.97 0.06 −0.22 0.13 No No F590A −0.43 0.05 −1.38 0.07 −1.070.15 No No F590R −0.22 0.05 −0.83 0.05 −0.62 0.15 No No F590N −0.95 0.17−0.65 0.15 NA NA Yes No F590C −0.49 0.08 −0.73 0.08 −1.06 0.20 No NoF590E −1.00 0.09 −1.26 0.10 −0.44 0.26 No No F590G −1.05 0.04 −1.44 0.05−1.47 0.09 No No F590I 0.01 0.09 0.10 0.08 NA NA Yes No F590L −0.02 0.04−0.21 0.04 −0.51 0.06 No No F590M −0.27 0.12 −0.41 0.12 NA NA Yes NoF590P −1.08 0.10 −1.31 0.10 −1.37 0.17 No No F590S −0.03 0.05 −0.29 0.05−0.61 0.07 No No F590T −1.50 0.15 −1.08 0.12 NA NA Yes No F590W 0.040.07 −0.11 0.07 −0.87 0.26 Yes No F590Y 0.07 0.06 −0.18 0.07 −0.19 0.17Yes No F590V −1.03 0.06 −0.97 0.06 −0.98 0.14 No No D591A 0.20 0.04 0.070.04 −0.54 0.05 Yes No D591R −0.94 0.05 −1.06 0.05 −0.45 0.13 No NoD591N −0.49 0.10 −0.61 0.10 −0.54 0.14 No No D591C −0.66 0.10 −0.68 0.09−0.47 0.23 No No D591Q −0.34 0.10 0.11 0.09 −0.15 0.38 Yes No D591E−0.43 0.05 −0.39 0.05 −0.84 0.14 No No D591G −0.62 0.03 −0.81 0.03 −0.590.03 No No D591H −1.19 0.16 −0.84 0.14 NA NA Yes No D591L −0.17 0.06−0.59 0.07 −0.99 0.20 No No D591K −0.51 0.12 −0.27 0.11 NA NA Yes NoD591M −0.90 0.12 −0.93 0.12 −0.36 0.27 No No D591S −0.69 0.07 −1.41 0.09−0.67 0.17 No No D591T −0.15 0.08 −0.93 0.10 −1.04 0.23 No No D591W−1.02 0.08 −0.91 0.07 −0.42 0.16 No No D591Y 0.08 0.10 −0.62 0.12 −0.500.17 Yes No D591V −0.76 0.05 −1.11 0.05 −1.12 0.11 No No K592A −1.600.09 −0.60 0.06 −1.42 0.15 No No K592R −0.98 0.05 −0.77 0.04 −1.03 0.05No No K592N −0.12 0.10 0.07 0.10 −0.91 0.19 Yes No K592Q −0.52 0.07−0.77 0.08 −0.30 0.10 No No K592E −0.52 0.07 −0.76 0.07 −1.22 0.10 No NoK592G −1.76 0.06 −1.45 0.05 −1.64 0.08 No No K592L −1.72 0.08 −1.52 0.08−1.24 0.13 No No K592M −0.70 0.07 −0.58 0.07 −0.68 0.07 No No K592P−1.70 0.14 −1.63 0.13 NA NA Yes No K592S −1.67 0.10 −1.56 0.10 NA NA YesNo K592* 0.00 0.08 −1.63 0.13 −0.91 0.18 Yes No K592T −1.38 0.11 −0.980.09 NA NA Yes No K592W −1.60 0.10 −1.14 0.08 −0.21 0.20 No No K592V−1.60 0.06 −1.14 0.05 −1.90 0.08 No No M593A −1.94 0.07 −1.00 0.05 −1.730.06 No No M593R −0.79 0.03 −0.81 0.03 −0.81 0.03 No No M593N −1.58 0.13−1.89 0.15 NA NA Yes No M593D −2.12 0.13 −1.32 0.09 −1.77 0.18 No NoM593C −0.95 0.07 −0.04 0.06 −1.27 0.10 No No M593Q NA NA NA NA −0.830.19 Yes No M593E −2.30 0.10 −2.14 0.09 −1.55 0.14 No No M593G −1.830.04 −1.88 0.04 −1.09 0.07 No No M593I −0.83 0.07 −1.05 0.08 −1.17 0.12No No M593L −0.10 0.03 −0.33 0.03 −0.52 0.06 No No M593K −1.64 0.09−1.56 0.08 −1.09 0.05 No No M593F −1.98 0.12 −0.83 0.08 −1.61 0.12 No NoM593P −1.56 0.10 −2.23 0.12 −0.38 0.12 No No M593S −1.58 0.06 −1.88 0.07−1.33 0.11 No No M593* −2.27 0.12 −2.28 0.12 −1.55 0.18 No No M593T−1.01 0.06 −0.79 0.05 −0.64 0.10 No No M593W −0.92 0.05 −1.57 0.07 −2.410.16 No No M593V −0.56 0.03 −1.41 0.04 −1.07 0.07 No No Y594A −0.40 0.04−1.29 0.05 −1.34 0.10 No No Y594R −0.02 0.03 −0.18 0.03 −1.14 0.08 No NoY594N −1.08 0.10 −0.80 0.08 −0.62 0.15 No No Y594D −1.17 0.07 −1.36 0.07−1.43 0.13 No No Y594C −0.47 0.06 −0.08 0.06 −1.45 0.10 No No Y594Q−1.06 0.08 −0.26 0.06 −1.14 0.12 No No Y594E −0.42 0.04 −0.82 0.05 −2.580.11 No No Y594G −1.21 0.03 −1.58 0.03 −1.51 0.04 No No Y594H 0.04 0.07−0.11 0.07 −0.42 0.12 Yes No Y594I −0.73 0.10 −0.78 0.10 −0.74 0.27 NoNo Y594L −0.88 0.05 −0.67 0.04 −1.45 0.10 No No Y594K −0.40 0.06 −0.880.07 −1.41 0.17 No No Y594M 0.05 0.06 −0.76 0.07 −1.47 0.18 Yes No Y594F−0.18 0.07 −0.07 0.06 −0.45 0.14 No No Y594S −1.00 0.05 −0.88 0.05 −1.400.06 No No Y594* −1.41 0.08 −0.73 0.06 −1.67 0.13 No No Y594T −1.66 0.09−1.71 0.09 −1.37 0.15 No No Y594W −0.33 0.04 −0.93 0.05 −1.06 0.10 No NoY594V −0.40 0.04 −0.47 0.04 −0.96 0.08 No No Y595A −1.63 0.05 −1.08 0.04−2.32 0.10 No No Y595R −1.29 0.04 −1.66 0.05 −2.00 0.06 No No Y595N−1.61 0.14 −0.78 0.10 −0.80 0.19 No No Y595D −1.27 0.09 −1.50 0.09 −1.840.06 No No Y595C −0.14 0.05 −0.50 0.05 −1.06 0.11 No No Y595Q −1.71 0.10−1.18 0.08 −1.13 0.22 No No Y595E −2.05 0.08 −2.21 0.08 −2.32 0.03 No NoY595G −1.67 0.03 −1.31 0.03 −1.77 0.03 No No Y595H −0.86 0.09 −0.57 0.08−0.77 0.13 No No Y595I −1.20 0.14 −1.49 0.15 NA NA Yes No Y595L −0.090.04 0.08 0.04 −0.83 0.12 Yes No Y595K −1.17 0.09 −1.74 0.11 NA NA YesNo Y595M 0.24 0.06 −0.10 0.07 −0.34 0.19 Yes No Y595F −1.21 0.09 −0.700.08 −1.09 0.14 No No Y595S −1.31 0.05 −1.19 0.05 −1.64 0.09 No No Y595*−1.32 0.07 −1.52 0.08 −1.60 0.09 No No Y595T −0.27 0.05 −0.81 0.06 NA NAYes No Y595W −1.72 0.06 −1.71 0.06 −1.97 0.10 No No Y595V −0.32 0.03−0.74 0.04 −2.00 0.06 No No D596E 0.04 0.04 0.24 0.04 0.69 0.05 Yes YesD596A 0.07 0.03 −0.18 0.04 −0.99 0.09 Yes No D596R 0.01 0.03 0.07 0.03−0.20 0.04 Yes No D596N −0.12 0.08 0.29 0.08 0.39 0.15 Yes No D596C−0.01 0.07 −0.24 0.07 −0.55 0.06 No No D596Q −0.04 0.09 0.42 0.08 0.460.31 Yes No D596G −0.23 0.02 −0.22 0.02 −1.16 0.05 No No D596H 0.24 0.10−0.18 0.11 0.16 0.26 Yes No D596L −0.92 0.07 −0.10 0.05 −0.75 0.18 No NoD596K 0.01 0.08 0.36 0.07 −0.07 0.19 Yes No D596M 0.26 0.08 0.29 0.07−0.27 0.28 Yes No D596F 0.03 0.11 −0.55 0.13 0.09 0.13 Yes No D596P NANA NA NA −0.56 0.24 Yes No D596S −0.58 0.06 −0.29 0.05 0.37 0.07 Yes NoD596* −0.99 0.11 −1.29 0.12 NA NA Yes No D596T −1.56 0.12 0.65 0.06−1.10 0.23 Yes No D596W −0.19 0.05 0.12 0.05 −1.34 0.11 Yes No D596Y−0.55 0.09 −0.32 0.08 −0.27 0.16 No No D596V −0.06 0.03 −0.22 0.03 −1.390.08 No No Y597A −2.00 0.06 −1.33 0.05 −2.10 0.12 No No Y597R −2.18 0.05−2.13 0.05 −2.18 0.04 No No Y597N −1.28 0.11 −0.66 0.09 −1.35 0.13 No NoY597D −0.30 0.03 −0.28 0.03 −0.34 0.04 No No Y597C −0.79 0.06 −1.14 0.07−1.47 0.05 No No Y597E −2.33 0.09 −2.41 0.09 NA NA Yes No Y597G −1.890.04 −2.15 0.04 −1.96 0.05 No No Y597H −0.45 0.07 −0.25 0.06 −0.71 0.10No No Y597I −0.51 0.11 −1.26 0.13 NA NA Yes No Y597L 0.24 0.04 0.32 0.04−0.31 0.09 Yes No Y597M −0.11 0.07 −1.15 0.10 NA NA Yes No Y597F −0.080.07 −0.16 0.07 0.56 0.06 Yes No Y597P −2.03 0.11 −2.30 0.11 NA NA YesNo Y597S −1.25 0.05 −1.04 0.05 −2.00 0.09 No No Y597* −0.94 0.06 −0.960.06 −2.02 0.13 No No Y597T −1.78 0.09 −2.62 0.12 −1.94 0.04 No No Y597W−1.16 0.05 −1.33 0.05 −2.30 0.08 No No Y597V −1.95 0.07 −1.63 0.06 −2.690.08 No No F598A −1.68 0.07 −1.98 0.08 −2.09 0.12 No No F598R −1.81 0.06−2.21 0.07 −1.57 0.11 No No F598C −1.96 0.12 −2.25 0.13 −1.21 0.15 No NoF598G −2.06 0.05 −1.79 0.04 −0.96 0.03 No No F598I −0.32 0.09 −0.44 0.09−0.75 0.12 No No F598L −0.25 0.04 −0.09 0.04 −0.65 0.04 No No F598M−0.35 0.09 −0.55 0.09 −0.87 0.27 No No F598P NA NA NA NA −1.93 0.07 YesNo F598S −1.12 0.06 −1.33 0.06 −1.35 0.08 No No F598W −1.23 0.06 −0.340.05 −1.74 0.12 No No F598Y −0.52 0.09 −0.44 0.09 −0.60 0.15 No No F598V−1.37 0.06 −1.39 0.05 −2.25 0.09 No No P599G 0.84 0.04 0.83 0.04 1.010.05 Yes Yes P599A −1.03 0.07 −0.85 0.06 −1.42 0.11 No No P599R −1.890.09 −1.20 0.06 −2.08 0.14 No No P599N −0.66 0.16 −0.33 0.14 NA NA YesNo P599E −2.04 0.15 −2.07 0.15 NA NA Yes No P599H −1.01 0.14 −0.99 0.14NA NA Yes No P599L −1.56 0.09 −1.36 0.08 −1.46 0.11 No No P599S −0.090.03 −0.10 0.03 −0.56 0.04 No No P599* −1.14 0.13 −1.42 0.14 NA NA YesNo P599T −0.42 0.07 −0.95 0.08 −1.37 0.14 No No P599V −1.88 0.12 −1.850.11 −1.81 0.14 No No D600A 0.43 0.06 −0.31 0.07 0.68 0.14 Yes No D600R−0.88 0.07 −0.40 0.06 −1.67 0.19 No No D600N −0.03 0.07 −0.16 0.07 0.670.14 Yes No D600C NA NA NA NA −0.32 0.48 Yes No D600Q −0.37 0.15 −0.010.13 NA NA Yes No D600E −0.44 0.07 −0.46 0.07 −0.95 0.22 No No D600G−0.19 0.04 −0.33 0.04 1.07 0.05 Yes No D600H −0.08 0.13 −0.54 0.14 NA NAYes No D600L −1.26 0.13 −1.19 0.12 −0.50 0.29 No No D600P −0.14 0.11−1.46 0.16 0.47 0.45 Yes No D600S 0.86 0.07 −0.03 0.08 0.74 0.14 Yes NoD600T −1.42 0.15 −0.70 0.12 0.43 0.29 Yes No D600W −0.67 0.11 −0.51 0.10NA NA Yes No D600Y −0.29 0.13 −0.19 0.12 NA NA Yes No D600V −0.05 0.06−0.23 0.06 −0.56 0.13 No No A601R −1.65 0.07 −1.62 0.07 NA NA Yes NoA601D −1.38 0.13 −1.10 0.11 NA NA Yes No A601C 0.26 0.08 0.26 0.08 −0.250.24 Yes No A601G −1.05 0.04 −0.63 0.04 −1.44 0.04 No No A601L −2.160.13 −1.44 0.09 NA NA Yes No A601P −1.40 0.13 −1.20 0.11 NA NA Yes NoA601S −0.60 0.06 0.23 0.04 −0.52 0.08 Yes No A601T −0.84 0.08 −0.53 0.07−0.99 0.12 No No A601W −1.89 0.12 −1.84 0.11 NA NA Yes No A601V −0.480.05 −0.23 0.04 −0.77 0.06 No No A602C 0.64 0.09 0.61 0.09 0.73 0.28 YesYes A602R −0.59 0.07 −0.97 0.08 NA NA Yes No A602D 0.04 0.04 −0.09 0.04−0.40 0.05 Yes No A602G −0.52 0.05 −0.56 0.05 −1.62 0.06 No No A602H NANA NA NA 0.37 0.35 Yes No A602L −1.97 0.14 −1.06 0.10 NA NA Yes No A602P−0.80 0.09 −0.42 0.08 −0.71 0.15 No No A602S −0.38 0.07 −0.14 0.06 −0.730.16 No No A602T −0.28 0.06 −0.03 0.06 −0.51 0.11 No No A602W NA NA NANA −0.89 0.37 Yes No A602V −0.77 0.06 −0.66 0.05 −1.41 0.10 No No K603A−1.34 0.07 −1.59 0.07 −1.76 0.05 No No K603R −0.56 0.04 −0.37 0.03 −0.460.04 No No K603N −1.59 0.16 −1.11 0.13 NA NA Yes No K603C −1.03 0.10−1.23 0.10 NA NA Yes No K603Q −1.30 0.11 −1.57 0.12 NA NA Yes No K603E−1.40 0.07 −0.68 0.05 −1.60 0.10 No No K603G −1.88 0.05 −1.70 0.04 −1.970.08 No No K603H NA NA NA NA −0.51 0.41 Yes No K603L −0.77 0.06 −0.600.06 −1.63 0.14 No No K603M 0.00 0.06 −0.57 0.07 −1.02 0.11 No No K603P−1.70 0.12 −1.06 0.09 NA NA Yes No K603S NA NA NA NA −2.06 0.05 Yes NoK603* −1.20 0.09 −1.66 0.11 −1.37 0.16 No No K603T −1.11 0.08 −1.15 0.08−1.13 0.13 No No K603W −2.79 0.14 −1.74 0.09 −1.45 0.14 No No K603V−1.72 0.07 −1.54 0.06 −2.40 0.07 No No M604A −2.19 0.15 −1.92 0.13 NA NAYes No M604R −0.96 0.06 −1.76 0.08 −2.02 0.13 No No M604G −2.12 0.08−0.82 0.05 −1.81 0.12 No No M604I −0.58 0.10 −0.71 0.10 −0.51 0.15 No NoM604L −0.74 0.06 −0.50 0.06 −0.25 0.06 No No M604K −1.08 0.15 −0.78 0.13NA NA Yes No M604T −0.14 0.06 0.01 0.06 −0.44 0.09 Yes No M604W −1.970.14 −2.03 0.14 NA NA Yes No M604V −1.38 0.08 −1.32 0.07 −1.38 0.09 NoNo I605A −1.32 0.09 −1.04 0.08 NA NA Yes No I605R −2.07 0.09 −2.36 0.10−2.12 0.15 No No I605N −0.93 0.10 −0.19 0.08 −0.76 0.16 No No I605C 0.200.09 −0.98 0.13 NA NA Yes No I605G −1.69 0.07 −1.41 0.06 −2.04 0.14 NoNo I605L −0.93 0.07 −0.25 0.06 −1.49 0.14 No No I605M −1.09 0.09 −0.860.08 −1.44 0.15 No No I605F −0.50 0.11 −0.52 0.10 NA NA Yes No I605S−1.91 0.11 −1.82 0.10 −1.55 0.10 No No I605T −0.45 0.07 −0.67 0.07 −1.090.12 No No I605V 0.13 0.05 −0.04 0.05 −0.33 0.11 Yes No I605A −1.42 0.10−0.82 0.08 NA NA Yes No I605R −2.12 0.09 −2.15 0.10 −1.82 0.16 No NoI605N 0.04 0.06 0.36 0.06 −0.41 0.09 Yes No I605D −1.00 0.09 −0.60 0.08−0.72 0.14 No No I605C 0.10 0.10 −0.82 0.13 NA NA Yes No I605G −1.690.07 −1.23 0.06 −1.96 0.13 No No I605L −0.10 0.03 0.14 0.03 −1.00 0.08Yes No I605M −0.89 0.09 −0.63 0.08 −1.25 0.21 No No I605F −0.55 0.10−0.33 0.09 NA NA Yes No I605S −0.22 0.04 0.10 0.04 −1.29 0.08 Yes NoI605T −0.02 0.04 0.21 0.04 −0.53 0.07 Yes No I605V 0.19 0.05 0.25 0.05−0.06 0.10 Yes No P606A −1.59 0.10 −2.27 0.12 −1.25 0.17 No No P606R−1.77 0.08 −2.62 0.12 −1.94 0.15 No No P606G −1.63 0.07 −2.12 0.08 −1.890.14 No No P606H −0.89 0.13 −0.93 0.13 NA NA Yes No P606L −0.77 0.07−1.12 0.08 −1.30 0.15 No No P606S −1.42 0.10 −1.22 0.09 −1.16 0.14 No NoP606T −0.06 0.06 −0.63 0.07 −0.40 0.08 No No P606W −0.88 0.11 −1.05 0.11NA NA Yes No P606V NA NA NA NA −1.59 0.24 Yes No P606A −1.59 0.10 −2.270.14 −1.33 0.19 No No P606R −1.77 0.09 −1.59 0.08 −1.76 0.15 No No P606QNA NA NA NA −0.43 0.12 Yes No P606G −1.73 0.08 −2.00 0.09 −1.79 0.16 NoNo P606H 0.13 0.07 0.17 0.07 −0.14 0.04 Yes No P606L −0.98 0.08 −1.230.09 −1.00 0.16 No No P606S −1.64 0.12 −1.35 0.11 −1.37 0.17 No No P606T−0.10 0.07 −0.63 0.08 −0.65 0.11 No No P606W −1.06 0.12 −0.93 0.11 NA NAYes No P606V NA NA NA NA −1.33 0.27 Yes No K607A −1.61 0.05 −1.70 0.06−2.05 0.10 No No K607R −1.65 0.04 −1.32 0.03 −2.05 0.03 No No K607N−0.51 0.07 −0.19 0.07 NA NA Yes No K607D −2.48 0.14 −1.83 0.11 NA NA YesNo K607C −1.09 0.07 −1.20 0.08 NA NA Yes No K607Q −1.77 0.09 −1.64 0.09−1.87 0.13 No No K607E −1.93 0.06 −1.40 0.05 −1.61 0.07 No No K607G−1.81 0.03 −1.60 0.03 −1.79 0.03 No No K607H −1.16 0.12 −0.39 0.10 NA NAYes No K607L −1.08 0.05 −1.66 0.06 −1.63 0.08 No No K607M −0.42 0.06−1.47 0.08 −0.97 0.05 No No K607F −1.55 0.12 −1.20 0.11 NA NA Yes NoK607P NA NA NA NA −1.84 0.14 Yes No K607S −1.89 0.06 −1.43 0.06 −1.530.04 No No K607* −1.44 0.07 −0.97 0.06 −1.20 0.14 No No K607T −2.23 0.09−1.26 0.06 −1.98 0.11 No No K607W −1.92 0.06 −1.88 0.06 −1.81 0.10 No NoK607V −1.80 0.05 −1.85 0.06 −1.86 0.03 No No C608A −1.60 0.09 −1.32 0.08−1.81 0.15 No No C608R −0.90 0.04 −0.72 0.04 −1.23 0.09 No No C608E−1.06 0.09 −1.27 0.10 NA NA Yes No C608G −1.44 0.05 −0.58 0.04 −1.660.09 No No C608L −1.11 0.08 −0.49 0.07 NA NA Yes No C608F −1.13 0.12−0.91 0.12 NA NA Yes No C608P −1.62 0.14 −1.13 0.12 NA NA Yes No C608S−0.62 0.05 −0.49 0.05 −1.56 0.12 No No C608* −1.61 0.13 −0.66 0.10 NA NAYes No C608W −1.58 0.09 −1.31 0.08 NA NA Yes No C608Y −0.27 0.06 −0.090.06 −0.65 0.15 No No C608V −1.32 0.07 −2.23 0.11 NA NA Yes No S609A0.10 0.07 0.21 0.07 −0.16 0.22 Yes No S609R −0.60 0.05 −0.45 0.05 −1.320.06 No No S609N −1.18 0.12 −0.49 0.10 NA NA Yes No S609C −0.69 0.120.21 0.09 −0.27 0.25 Yes No S609G 0.00 0.04 −0.27 0.04 −0.17 0.10 No NoS609I −0.16 0.12 0.43 0.11 NA NA Yes No S609T −0.76 0.12 0.55 0.09 NA NAYes No S609W −1.36 0.13 −0.51 0.10 NA NA Yes No T610A −0.40 0.05 −0.400.05 −0.93 0.09 No No T610R −1.61 0.07 −0.98 0.06 −1.76 0.15 No No T610N−0.27 0.10 −0.20 0.10 −0.58 0.12 No No T610C −0.26 0.09 −0.31 0.09 NA NAYes No T610Q NA NA NA NA −0.53 0.36 Yes No T610G −1.36 0.05 −1.25 0.05−1.62 0.12 No No T610I −0.82 0.11 −0.98 0.12 NA NA Yes No T610L −1.780.11 −2.29 0.14 NA NA Yes No T610P 0.07 0.04 0.33 0.04 −0.62 0.08 Yes NoT610S −0.35 0.06 −0.62 0.06 −0.53 0.07 No No T610V 0.26 0.05 −1.14 0.08−1.58 0.17 Yes No Q611A −2.61 0.13 −1.23 0.08 −1.40 0.13 No No Q611R−0.68 0.04 −0.69 0.04 −1.33 0.04 No No Q611E −1.22 0.07 −0.58 0.06 −1.180.11 No No Q611G −1.38 0.05 −1.84 0.06 −1.82 0.08 No No Q611H 0.04 0.07−0.20 0.07 −0.91 0.13 Yes No Q611L −1.21 0.08 −1.74 0.10 −1.67 0.16 NoNo Q611K −0.93 0.09 −0.83 0.09 −1.38 0.16 No No Q611P 0.13 0.06 −0.140.06 −0.81 0.08 Yes No Q611S −2.07 0.11 −0.89 0.07 NA NA Yes No Q611*−0.75 0.09 −1.11 0.10 −1.17 0.16 No No Q611W −1.28 0.08 −0.88 0.07 NA NAYes No Q611V −1.42 0.07 −1.15 0.07 NA NA Yes No L612M 0.70 0.07 0.700.07 0.01 0.03 Yes Yes L612A −1.32 0.11 −1.11 0.11 −0.98 0.22 No NoL612R 0.03 0.05 0.16 0.05 −0.58 0.11 Yes No L612Q −0.94 0.10 −0.43 0.09NA NA Yes No L612E −1.18 0.11 −1.65 0.13 NA NA Yes No L612G −0.97 0.07−2.15 0.11 −1.70 0.17 No No L612I −0.18 0.13 0.66 0.11 −0.24 0.15 Yes NoL612K −1.41 0.16 −0.48 0.12 NA NA Yes No L612P −0.34 0.06 −0.33 0.06−0.72 0.11 No No L612* −1.69 0.15 −0.55 0.10 NA NA Yes No L612T −0.270.09 −1.35 0.13 NA NA Yes No L612V −1.05 0.08 −0.90 0.08 −1.51 0.05 NoNo K613R −0.22 0.06 −0.18 0.06 −0.40 0.05 No No K613N 0.04 0.10 0.160.10 NA NA Yes No K613Q 0.14 0.04 0.38 0.04 NA NA Yes No K613E −0.750.08 0.06 0.07 NA NA Yes No K613G −1.17 0.08 −1.36 0.09 NA NA Yes NoK613L 0.12 0.09 0.49 0.09 −0.52 0.26 Yes No K613M −0.26 0.11 0.73 0.09NA NA Yes No K613* −0.72 0.11 −0.72 0.11 NA NA Yes No K613T 0.26 0.050.46 0.05 −0.65 0.08 Yes No K613W −1.27 0.12 −1.60 0.14 NA NA Yes NoK613V −0.89 0.10 −0.25 0.08 NA NA Yes No A614R 0.28 0.04 0.07 0.05 0.160.05 Yes Yes A614I 0.52 0.11 0.59 0.11 0.41 0.22 Yes Yes A614D −0.020.08 −0.34 0.09 NA NA Yes No A614C −0.54 0.09 −0.12 0.08 0.02 0.22 YesNo A614Q 0.50 0.10 0.17 0.11 NA NA Yes No A614E −0.71 0.08 −0.80 0.09 NANA Yes No A614G −0.16 0.03 0.09 0.03 −0.21 0.08 Yes No A614L 0.09 0.06−0.21 0.06 0.02 0.12 Yes No A614K NA NA NA NA 0.28 0.38 Yes No A614M−0.39 0.13 1.39 0.09 NA NA Yes No A614F −0.66 0.14 −0.11 0.12 NA NA YesNo A614P −0.22 0.09 −0.09 0.09 −0.68 0.25 No No A614S −0.46 0.07 0.190.06 −0.55 0.15 Yes No A614* NA NA NA NA −0.35 0.32 Yes No A614T 0.030.06 0.37 0.06 −0.49 0.14 Yes No A614W −0.74 0.07 −0.88 0.08 −0.97 0.14No No A614V 0.17 0.04 0.29 0.04 −0.03 0.11 Yes No V615A −0.05 0.03 0.090.03 −0.35 0.04 Yes No V615R −1.73 0.05 −1.23 0.04 −1.21 0.07 No NoV615N −0.21 0.08 −1.01 0.11 −0.72 0.15 No No V615D −0.95 0.07 −0.97 0.07−1.54 0.13 No No V615C −0.18 0.06 −0.18 0.06 −0.01 0.19 No No V615Q−1.87 0.10 −1.41 0.09 NA NA Yes No V615E −0.82 0.04 −0.59 0.04 −1.030.05 No No V615G −0.57 0.03 −0.88 0.03 −1.19 0.04 No No V615I −0.51 0.09−0.73 0.10 −0.67 0.23 No No V615L −0.07 0.03 0.05 0.03 −0.88 0.05 Yes NoV615K −1.22 0.08 −1.81 0.10 −1.56 0.07 No No V615M −0.52 0.06 −0.54 0.06−0.54 0.12 No No V615F −1.88 0.14 −1.30 0.12 NA NA Yes No V615P −1.530.10 −1.82 0.11 −1.42 0.06 No No V615S 0.17 0.04 −0.23 0.04 −0.63 0.05Yes No V615* −1.04 0.07 −0.78 0.07 −1.35 0.16 No No V615T 0.19 0.05−0.12 0.06 −0.07 0.17 Yes No V615W −1.19 0.06 −1.55 0.07 −1.90 0.05 NoNo T616A 0.32 0.03 0.24 0.04 0.09 0.05 Yes Yes T616R 0.19 0.03 0.12 0.030.62 0.04 Yes Yes T616Q 0.35 0.07 0.38 0.07 0.78 0.20 Yes Yes T616G 0.220.03 0.01 0.03 0.16 0.06 Yes Yes T616Y 0.54 0.09 0.64 0.09 0.05 0.38 YesYes T616N −0.11 0.08 0.20 0.08 0.16 0.07 Yes No T616D −0.66 0.08 0.140.07 −0.18 0.26 Yes No T616C 0.54 0.06 0.27 0.06 −0.24 0.11 Yes No T616E−0.28 0.06 −0.38 0.06 −0.17 0.07 No No T616H 0.62 0.08 −0.13 0.10 1.210.25 Yes No T616I −0.33 0.08 0.30 0.07 0.19 0.18 Yes No T616L −0.27 0.05−0.43 0.05 −0.19 0.13 No No T616K −0.36 0.08 0.23 0.07 0.66 0.09 Yes NoT616M −0.49 0.08 −0.40 0.08 −0.44 0.25 No No T616F −0.58 0.11 0.07 0.090.10 0.36 Yes No T616P −0.89 0.07 −0.68 0.07 −0.80 0.06 No No T616S−0.02 0.04 0.12 0.04 0.13 0.07 Yes No T616* NA NA NA NA −1.14 0.24 YesNo T616W −0.28 0.05 0.25 0.04 −0.22 0.19 Yes No T616V −0.07 0.04 −0.090.04 0.21 0.07 Yes No A617G 0.03 0.03 0.05 0.03 0.24 0.06 Yes Yes A617R−0.10 0.04 0.07 0.04 −0.32 0.11 Yes No A617N NA NA NA NA 0.12 0.42 YesNo A617D −0.76 0.09 −0.05 0.07 NA NA Yes No A617C −0.73 0.09 0.26 0.070.13 0.18 Yes No A617Q 0.25 0.08 0.07 0.09 −0.48 0.30 Yes No A617E −0.120.06 0.08 0.06 −0.40 0.20 Yes No A617H −0.11 0.11 −0.46 0.12 NA NA YesNo A617I −0.26 0.11 0.09 0.10 −0.52 0.38 Yes No A617L −0.11 0.05 0.180.05 −0.18 0.16 Yes No A617K NA NA NA NA −0.08 0.28 Yes No A617M 0.320.07 0.30 0.07 −0.34 0.27 Yes No A617F −0.17 0.10 −0.39 0.11 −0.42 0.36No No A617P −0.48 0.08 −0.88 0.09 0.05 0.18 Yes No A617S −0.13 0.05 0.250.05 −0.58 0.06 Yes No A617* −0.96 0.11 −1.57 0.14 NA NA Yes No A617T−0.23 0.05 0.26 0.05 −0.30 0.10 Yes No A617W 0.17 0.05 −0.93 0.07 −0.690.15 Yes No A617V −0.03 0.04 −0.09 0.04 −0.42 0.12 No No H618A −0.730.14 −0.80 0.15 0.36 0.36 Yes No H618R −0.08 0.05 0.24 0.05 −0.46 0.06Yes No H618N −0.37 0.17 −0.24 0.16 NA NA Yes No H618D 0.48 0.13 0.890.12 NA NA Yes No H618Q −0.62 0.14 0.01 0.12 NA NA Yes No H618G −0.560.07 −0.91 0.09 −0.91 0.19 No No H618L 0.02 0.06 0.10 0.06 −0.47 0.07Yes No H618P 0.32 0.08 0.47 0.08 −0.51 0.08 Yes No H618S −0.50 0.14−0.26 0.13 NA NA Yes No H618W NA NA NA NA 0.44 0.31 Yes No H618Y 0.230.10 −0.28 0.11 −0.26 0.19 Yes No F619M 0.12 0.07 0.55 0.07 0.22 0.25Yes Yes F619A −0.57 0.05 −1.25 0.06 −0.78 0.05 No No F619R −1.24 0.05−1.94 0.07 −0.66 0.07 No No F619N −0.61 0.13 −0.15 0.12 NA NA Yes NoF619D −1.39 0.12 −0.62 0.09 NA NA Yes No F619C 0.07 0.03 0.08 0.03 −0.370.04 Yes No F619Q −1.13 0.12 −1.57 0.15 NA NA Yes No F619E −1.58 0.08−2.40 0.12 −1.54 0.08 No No F619G −1.29 0.03 −1.03 0.03 −1.57 0.05 No NoF619H NA NA NA NA −0.31 0.34 Yes No F619I −0.69 0.12 −0.28 0.11 −0.420.22 No No F619L 0.17 0.03 0.12 0.03 −0.48 0.05 Yes No F619P −1.00 0.09−1.22 0.10 NA NA Yes No F619S −0.26 0.04 −0.95 0.06 −0.56 0.06 No NoF619* −1.91 0.14 −0.92 0.10 NA NA Yes No F619T NA NA NA NA −1.08 0.24Yes No F619W −0.08 0.04 −0.81 0.05 0.00 0.07 Yes No F619Y 0.06 0.08−0.79 0.11 0.28 0.11 Yes No F619V −0.51 0.04 −0.49 0.04 −0.64 0.04 No NoQ620A 0.22 0.04 0.00 0.05 0.18 0.09 Yes Yes Q620R 0.25 0.03 0.18 0.030.17 0.04 Yes Yes Q620N 0.26 0.14 0.28 0.14 0.36 0.21 Yes Yes Q620L 0.140.04 0.45 0.04 0.40 0.16 Yes Yes Q620K 0.10 0.07 0.24 0.07 0.04 0.10 YesYes Q620D −0.16 0.07 −0.25 0.08 0.24 0.23 Yes No Q620C −0.22 0.07 −0.950.10 0.03 0.23 Yes No Q620E 0.21 0.05 0.13 0.05 −0.09 0.10 Yes No Q620G−0.14 0.02 0.02 0.02 0.19 0.04 Yes No Q620H −0.08 0.08 0.16 0.08 −0.050.20 Yes No Q620I 0.27 0.11 −0.38 0.14 NA NA Yes No Q620M 0.70 0.07−0.03 0.08 0.20 0.26 Yes No Q620F 0.37 0.11 1.00 0.10 −0.47 0.44 Yes NoQ620P −0.04 0.06 0.12 0.06 −0.26 0.10 Yes No Q620S 0.17 0.05 −0.15 0.060.16 0.17 Yes No Q620* −0.82 0.08 −0.58 0.07 −0.64 0.15 No No Q620T−0.20 0.09 0.41 0.08 0.17 0.12 Yes No Q620W −0.12 0.04 0.09 0.04 0.080.11 Yes No Q620Y −0.74 0.14 0.21 0.11 −0.19 0.38 Yes No Q620V 0.29 0.03−0.11 0.04 0.26 0.07 Yes No T621A 0.18 0.04 0.13 0.04 0.17 0.10 Yes YesT621R −0.04 0.04 −0.45 0.05 −0.06 0.15 No No T621N −0.39 0.12 −0.09 0.110.02 0.21 Yes No T621D 0.12 0.12 0.26 0.12 −0.11 0.44 Yes No T621C 0.070.09 −0.29 0.10 NA NA Yes No T621Q −0.90 0.12 0.06 0.10 0.44 0.38 Yes NoT621E −0.29 0.07 0.13 0.06 −0.05 0.24 Yes No T621G −0.02 0.03 −0.21 0.040.48 0.04 Yes No T621H 0.33 0.15 0.72 0.14 NA NA Yes No T621I 0.36 0.08−0.09 0.10 0.21 0.20 Yes No T621L 0.16 0.06 −0.40 0.07 −0.30 0.10 Yes NoT621K −0.02 0.09 −0.09 0.09 −0.08 0.37 No No T621M 0.21 0.07 −0.15 0.081.17 0.09 Yes No T621F −0.61 0.16 −0.16 0.14 NA NA Yes No T621P 0.040.04 0.18 0.04 −0.38 0.06 Yes No T621S −0.06 0.06 −0.08 0.06 0.28 0.09Yes No T621W −0.21 0.06 0.08 0.06 0.12 0.17 Yes No T621Y −0.23 0.16 0.360.14 NA NA Yes No T621V −0.28 0.05 0.32 0.05 0.32 0.19 Yes No H622G 0.050.03 0.18 0.03 0.07 0.06 Yes Yes H622S 0.16 0.06 0.59 0.06 0.08 0.10 YesYes H622T 0.01 0.09 0.84 0.08 0.31 0.34 Yes Yes H622V 0.18 0.05 0.150.05 0.31 0.12 Yes Yes H622A −0.17 0.06 0.16 0.06 0.41 0.08 Yes No H622R−0.19 0.04 0.12 0.04 0.05 0.04 Yes No H622N −0.05 0.07 0.30 0.07 −0.110.12 Yes No H622D −0.84 0.11 0.63 0.07 0.48 0.10 Yes No H622C −0.50 0.09−0.10 0.08 0.11 0.11 Yes No H622Q −0.06 0.08 0.29 0.07 −0.14 0.05 Yes NoH622E −0.11 0.07 0.15 0.06 0.23 0.10 Yes No H622I −0.05 0.14 0.45 0.130.36 0.49 Yes No H622L −0.49 0.06 0.06 0.05 −0.14 0.11 Yes No H622K 0.290.10 1.39 0.08 0.00 0.28 Yes No H622M −0.07 0.11 −0.07 0.11 0.05 0.34Yes No H622F 0.14 0.11 −0.61 0.14 0.07 0.13 Yes No H622P 0.14 0.03 0.250.03 −0.44 0.04 Yes No H622* −1.50 0.13 −1.18 0.12 −0.74 0.16 No NoH622W 0.08 0.05 0.21 0.05 −0.08 0.19 Yes No H622Y 0.07 0.09 0.14 0.09−0.08 0.18 Yes No T623E 0.32 0.05 0.23 0.05 0.02 0.05 Yes Yes T623H 0.600.10 0.01 0.11 0.58 0.55 Yes Yes T623L 0.33 0.05 0.18 0.05 0.17 0.16 YesYes T623M 0.21 0.07 0.28 0.07 0.59 0.22 Yes Yes T623F 0.80 0.10 0.610.10 0.47 0.32 Yes Yes T623A −0.14 0.04 0.35 0.03 −0.04 0.07 Yes NoT623R −0.16 0.03 −0.40 0.04 0.15 0.09 Yes No T623N 0.02 0.09 0.19 0.09−0.28 0.16 Yes No T623D −0.69 0.09 0.41 0.07 −0.34 0.14 Yes No T623C−0.39 0.08 0.01 0.08 0.40 0.28 Yes No T623Q 0.23 0.07 0.38 0.07 −0.010.32 Yes No T623G −0.40 0.03 −0.09 0.03 −0.05 0.03 No No T623I −0.590.10 0.11 0.08 −0.13 0.13 Yes No T623K 1.10 0.07 −0.02 0.08 0.53 0.17Yes No T623P −0.16 0.06 −0.29 0.07 −0.11 0.17 No No T623S 0.67 0.04 0.210.04 −0.06 0.07 Yes No T623W −0.01 0.05 −0.29 0.06 −0.27 0.10 No NoT623Y NA NA NA NA −0.11 0.29 Yes No T623V −0.26 0.04 −0.20 0.04 0.410.05 Yes No T624P 0.92 0.02 0.92 0.02 0.04 0.03 Yes Yes T624A 0.65 0.020.76 0.02 −0.07 0.03 Yes No T624R 0.41 0.07 0.34 0.07 −0.55 0.23 Yes NoT624N 0.15 0.10 0.20 0.10 NA NA Yes No T624C −0.20 0.15 0.21 0.14 NA NAYes No T624E 0.38 0.09 −0.63 0.12 −0.11 0.55 Yes No T624G −0.21 0.060.12 0.06 0.15 0.21 Yes No T624I 0.58 0.11 0.35 0.11 NA NA Yes No T624L0.41 0.09 −0.84 0.13 0.96 0.48 Yes No T624S 1.15 0.04 1.20 0.04 −0.130.04 Yes No T624W 0.56 0.10 −0.20 0.12 0.10 0.16 Yes No T624V −0.12 0.08−0.52 0.10 0.18 0.23 Yes No P625A −0.27 0.11 0.30 0.10 0.31 0.10 Yes NoP625R 0.06 0.07 −1.01 0.10 −0.04 0.22 Yes No P625C 0.56 0.14 −0.17 0.16−0.02 0.82 Yes No P625E −0.24 0.13 −0.22 0.13 −0.41 0.64 No No P625G−0.41 0.08 0.40 0.06 0.04 0.24 Yes No P625H −0.25 0.15 −0.02 0.15 NA NAYes No P625L −0.48 0.09 −0.82 0.11 −0.56 0.17 No No P625S −0.38 0.08−0.55 0.09 0.03 0.20 Yes No P625T 0.30 0.08 0.23 0.08 −0.06 0.10 Yes NoP625W −0.60 0.13 −0.64 0.13 NA NA Yes No P625V −0.17 0.09 −0.63 0.11 NANA Yes No I626A 0.16 0.12 0.80 0.11 NA NA Yes No I626R −0.89 0.12 0.140.09 −0.09 0.31 Yes No I626N 0.42 0.04 0.61 0.04 −0.37 0.06 Yes No I626ENA NA NA NA −0.15 0.55 Yes No I626G −1.04 0.10 −1.35 0.12 −0.48 0.27 NoNo I626L 0.61 0.11 −0.85 0.16 NA NA Yes No I626S 0.36 0.04 0.60 0.04−0.65 0.07 Yes No I626T 0.17 0.02 0.34 0.02 −0.37 0.03 Yes No I626W−0.74 0.16 0.22 0.13 NA NA Yes No I626V −0.51 0.10 0.05 0.09 0.10 0.18Yes No L627C 0.14 0.13 0.84 0.11 0.57 0.48 Yes Yes L627A 0.00 0.08 0.240.08 1.31 0.29 Yes No L627R 0.29 0.04 0.03 0.04 −0.10 0.05 Yes No L627N0.71 0.15 1.09 0.15 NA NA Yes No L627D 0.32 0.10 −1.12 0.16 1.13 0.55Yes No L627Q 0.23 0.07 0.39 0.07 −0.27 0.11 Yes No L627E 0.29 0.07 −0.060.08 0.17 0.11 Yes No L627G −0.04 0.04 −0.01 0.04 0.23 0.10 Yes No L627K0.57 0.11 0.27 0.12 NA NA Yes No L627M −0.57 0.12 0.33 0.10 −0.09 0.22Yes No L627P 0.10 0.05 0.31 0.05 −0.25 0.08 Yes No L627S 0.34 0.08 0.180.08 −0.29 0.27 Yes No L627* 0.52 0.10 −0.87 0.15 NA NA Yes No L627T0.60 0.09 −0.76 0.14 NA NA Yes No L627W −0.71 0.09 0.85 0.07 0.27 0.32Yes No L627V −0.89 0.08 0.48 0.06 −0.23 0.19 Yes No L628C 0.31 0.09 0.100.10 0.29 0.31 Yes Yes L628W 0.25 0.06 0.01 0.07 0.67 0.15 Yes Yes L628A−0.22 0.06 −0.58 0.07 −0.36 0.11 No No L628R −0.13 0.04 −0.50 0.05 0.530.06 Yes No L628Q −0.10 0.10 −0.04 0.10 −0.36 0.23 No No L628E −0.730.10 −1.59 0.14 NA NA Yes No L628G −0.65 0.04 −1.57 0.06 −0.96 0.14 NoNo L628I −0.47 0.17 −0.57 0.17 NA NA Yes No L628K 0.75 0.11 −0.44 0.15−0.21 0.31 Yes No L628M −0.49 0.09 −0.37 0.09 −0.21 0.12 No No L628P−0.27 0.08 −0.45 0.08 −0.39 0.14 No No L628S −0.78 0.08 −1.36 0.10 −0.830.21 No No L628T −0.16 0.10 −0.52 0.12 −0.60 0.27 No No L628V −0.13 0.050.34 0.05 −0.31 0.09 Yes No S629A −0.99 0.12 0.49 0.08 −0.09 0.29 Yes NoS629R 0.10 0.02 0.29 0.02 −0.37 0.03 Yes No S629N 0.40 0.05 0.36 0.05−0.32 0.08 Yes No S629D −0.54 0.18 −0.23 0.17 NA NA Yes No S629C 0.300.10 0.10 0.11 −0.08 0.21 Yes No S629Q 0.76 0.17 0.40 0.18 NA NA Yes NoS629E 0.84 0.10 0.61 0.11 −0.08 0.48 Yes No S629G −0.30 0.05 0.40 0.050.09 0.08 Yes No S629I 0.05 0.14 0.42 0.13 NA NA Yes No S629L NA NA NANA 0.03 0.36 Yes No S629K 0.37 0.13 0.80 0.12 NA NA Yes No S629P −0.030.15 −0.34 0.17 NA NA Yes No S629T −0.60 0.13 0.01 0.11 0.22 0.32 Yes NoS629W −0.03 0.11 −0.66 0.13 0.42 0.54 Yes No S629V −0.87 0.11 −0.82 0.110.05 0.14 Yes No N630R 0.53 0.05 0.06 0.06 0.94 0.08 Yes Yes N630A −0.530.09 0.62 0.07 −0.06 0.23 Yes No N630D −0.33 0.09 0.49 0.07 −0.22 0.14Yes No N630C 0.42 0.10 −0.12 0.12 NA NA Yes No N630E −0.67 0.09 0.690.07 −0.30 0.26 Yes No N630G 0.32 0.04 −0.11 0.04 0.22 0.10 Yes No N630H0.06 0.10 0.51 0.09 −0.29 0.13 Yes No N630I −0.45 0.12 0.44 0.10 −0.270.18 Yes No N630L −0.24 0.09 0.55 0.08 −0.78 0.15 Yes No N630K 0.37 0.100.93 0.09 −0.25 0.18 Yes No N630F −0.58 0.18 −0.11 0.16 NA NA Yes NoN630S 0.34 0.06 −0.21 0.07 0.16 0.13 Yes No N630T 0.16 0.03 0.38 0.02−0.53 0.04 Yes No N630W −0.11 0.09 −1.04 0.12 NA NA Yes No N630Y −0.240.12 0.59 0.10 0.14 0.19 Yes No N630V −0.20 0.06 −0.90 0.08 −0.26 0.18No No N631A −0.03 0.07 0.06 0.07 −0.70 0.25 Yes No N631R −0.11 0.06 0.270.05 1.20 0.07 Yes No N631D −0.56 0.10 −0.38 0.09 −0.77 0.13 No No N631C−0.71 0.15 −0.64 0.15 NA NA Yes No N631E NA NA NA NA −0.95 0.23 Yes NoN631G −0.67 0.05 −0.39 0.05 −1.02 0.07 No No N631I 0.31 0.10 −0.62 0.13NA NA Yes No N631L −1.12 0.10 −0.57 0.08 NA NA Yes No N631K −0.05 0.08−0.20 0.09 0.70 0.11 Yes No N631M −0.10 0.10 0.36 0.09 −0.27 0.30 Yes NoN631P 0.06 0.12 0.30 0.12 NA NA Yes No N631S 0.15 0.06 0.06 0.06 −0.360.13 Yes No N631T 0.07 0.04 0.24 0.04 −0.37 0.05 Yes No N631Y −0.48 0.12−0.03 0.11 NA NA Yes No N631V −0.66 0.07 −1.17 0.08 −0.78 0.23 No NoF632A −0.79 0.07 −1.34 0.09 −1.29 0.12 No No F632R −0.76 0.05 −0.40 0.05−0.13 0.05 No No F632D −1.62 0.15 −1.61 0.15 NA NA Yes No F632C 0.660.02 0.78 0.02 −0.06 0.02 Yes No F632Q NA NA NA NA −0.18 0.55 Yes NoF632E −1.35 0.10 −0.87 0.09 NA NA Yes No F632G −0.01 0.03 0.12 0.03−0.03 0.02 Yes No F632H −0.31 0.16 0.08 0.15 NA NA Yes No F632I −0.330.10 −0.01 0.10 −0.87 0.13 No No F632L −0.28 0.05 0.22 0.05 −0.26 0.07Yes No F632K −0.49 0.13 0.74 0.10 NA NA Yes No F632M 0.00 0.10 0.16 0.10NA NA Yes No F632S −0.75 0.06 −0.56 0.06 −0.83 0.06 No No F632* −0.690.12 −0.55 0.12 NA NA Yes No F632T −0.09 0.09 −0.80 0.12 NA NA Yes NoF632W 0.17 0.06 −0.14 0.06 −0.44 0.19 Yes No F632Y −0.15 0.08 0.38 0.07−0.07 0.10 Yes No F632V 0.38 0.02 0.41 0.02 −0.07 0.02 Yes No I633N 0.160.06 0.65 0.06 0.15 0.10 Yes Yes I633M 0.17 0.07 0.28 0.07 0.10 0.19 YesYes I633S 0.02 0.04 0.35 0.04 0.22 0.08 Yes Yes I633A 0.15 0.05 −0.240.06 0.42 0.13 Yes No I633R −0.42 0.04 0.41 0.04 0.22 0.06 Yes No I633D−0.62 0.09 −0.01 0.08 −0.12 0.10 No No I633C 0.15 0.07 −0.34 0.08 −0.040.14 Yes No I633Q −0.26 0.10 1.05 0.08 −0.37 0.22 Yes No I633E −0.980.07 −0.34 0.06 0.30 0.11 Yes No I633G −0.09 0.03 0.10 0.03 0.05 0.06Yes No I633H −0.71 0.14 −0.16 0.13 NA NA Yes No I633L −0.10 0.05 0.000.05 0.25 0.06 Yes No I633K −0.29 0.09 0.17 0.08 0.63 0.29 Yes No I633F−0.11 0.09 0.24 0.09 0.51 0.22 Yes No I633P 0.23 0.08 −0.90 0.12 0.190.31 Yes No I633* −1.45 0.12 −1.53 0.13 NA NA Yes No I633T 0.13 0.040.40 0.04 −0.17 0.06 Yes No I633W −0.35 0.05 0.14 0.05 −0.07 0.09 Yes NoI633Y NA NA NA NA −0.09 0.34 Yes No I633V 0.09 0.04 −0.06 0.04 0.39 0.06Yes No E634N 0.52 0.12 0.46 0.12 0.00 0.36 Yes Yes E634A 0.20 0.02 0.380.02 −0.36 0.03 Yes No E634R −0.06 0.04 0.30 0.04 0.33 0.08 Yes No E634D−0.10 0.06 0.30 0.06 0.01 0.10 Yes No E634C −0.12 0.08 −0.30 0.09 0.420.13 Yes No E634Q −0.28 0.09 0.79 0.07 0.35 0.10 Yes No E634G −0.12 0.030.10 0.02 0.33 0.03 Yes No E634I 0.43 0.13 −0.05 0.15 NA NA Yes No E634L0.12 0.05 −0.46 0.06 0.47 0.19 Yes No E634K 0.14 0.06 −0.04 0.07 0.160.13 Yes No E634M −0.14 0.08 −0.22 0.08 0.42 0.27 Yes No E634F 0.25 0.140.08 0.15 NA NA Yes No E634P −0.23 0.10 −0.95 0.13 NA NA Yes No E634S−0.22 0.06 0.07 0.05 0.30 0.07 Yes No E634* 0.11 0.03 0.29 0.03 −0.340.05 Yes No E634T 0.01 0.09 0.30 0.08 −0.51 0.28 Yes No E634W −0.46 0.060.16 0.05 0.45 0.17 Yes No E634V −0.20 0.04 0.26 0.04 0.33 0.08 Yes NoP635A 0.04 0.05 0.29 0.05 0.59 0.07 Yes Yes P635D 0.14 0.09 0.49 0.090.52 0.14 Yes Yes P635E 0.51 0.06 0.49 0.06 0.70 0.16 Yes Yes P635T 0.360.06 0.06 0.06 0.01 0.08 Yes Yes P635R −0.51 0.04 −0.39 0.04 0.24 0.08Yes No P635N 0.13 0.14 −0.31 0.16 NA NA Yes No P635C −0.65 0.10 −0.700.11 0.26 0.25 Yes No P635Q −0.86 0.12 −0.40 0.11 0.43 0.38 Yes No P635G−0.01 0.03 −0.02 0.03 0.15 0.06 Yes No P635H −0.05 0.10 0.20 0.09 0.220.07 Yes No P635I NA NA NA NA 0.13 0.37 Yes No P635L −0.02 0.05 −0.330.06 0.22 0.10 Yes No P635K NA NA NA NA 0.18 0.41 Yes No P635M 0.96 0.08−0.61 0.12 0.06 0.31 Yes No P635F 1.00 0.11 0.14 0.13 NA NA Yes No P635S−0.22 0.06 0.35 0.05 0.02 0.12 Yes No P635W −0.24 0.06 0.25 0.06 −0.120.20 Yes No P635Y −0.31 0.15 −0.01 0.14 NA NA Yes No P635V −0.06 0.05−0.29 0.05 0.44 0.20 Yes No L636A −0.52 0.05 −1.17 0.06 0.08 0.11 Yes NoL636R −1.28 0.04 −1.29 0.04 −1.27 0.08 No No L636C −0.14 0.07 −0.19 0.07−0.07 0.17 No No L636Q −0.25 0.05 −0.09 0.05 −0.16 0.09 No No L636E−1.02 0.07 −0.96 0.07 −1.27 0.21 No No L636G −1.44 0.04 −1.31 0.04 −1.360.06 No No L636I −0.87 0.13 −0.24 0.11 NA NA Yes No L636K −0.17 0.08−0.33 0.09 −1.00 0.28 No No L636M −0.40 0.07 −0.08 0.06 0.16 0.16 Yes NoL636F −0.45 0.10 0.29 0.09 NA NA Yes No L636P 0.24 0.05 −0.84 0.06 −0.880.11 Yes No L636S −1.62 0.07 −1.47 0.07 −0.73 0.15 No No L636* −1.090.10 −1.51 0.12 NA NA Yes No L636T 0.12 0.06 −2.04 0.13 −0.41 0.09 YesNo L636W −0.55 0.05 −1.36 0.06 −0.68 0.11 No No L636V −0.35 0.04 −0.050.03 −0.65 0.08 No No E637A 0.15 0.04 0.23 0.04 −0.25 0.07 Yes No E637R−0.23 0.07 1.02 0.06 0.18 0.32 Yes No E637D −0.88 0.14 −0.01 0.11 NA NAYes No E637Q 0.83 0.14 0.47 0.15 NA NA Yes No E637G −0.05 0.04 0.22 0.040.06 0.05 Yes No E637L NA NA NA NA 0.35 0.32 Yes No E637K −0.33 0.12−0.66 0.13 NA NA Yes No E637M 0.60 0.15 1.04 0.14 NA NA Yes No E637S0.23 0.10 −0.55 0.12 0.66 0.39 Yes No E637* −0.03 0.09 0.24 0.08 NA NAYes No E637W −0.18 0.10 −0.73 0.12 −0.20 0.34 No No E637V −0.15 0.07−0.36 0.07 −0.45 0.10 No No I638R −1.56 0.13 −0.55 0.10 NA NA Yes NoI638N 0.35 0.16 0.42 0.16 −0.63 0.15 Yes No I638C 1.11 0.14 −0.05 0.17NA NA Yes No I638L NA NA NA NA −0.24 0.30 Yes No I638S 0.04 0.05 0.050.05 −0.18 0.05 Yes No I638T −0.43 0.12 −0.42 0.12 −0.14 0.18 No NoI638V 0.39 0.07 −0.45 0.08 −0.04 0.13 Yes No T639G 0.52 0.06 0.44 0.060.06 0.23 Yes Yes T639A 0.25 0.05 0.47 0.05 −0.34 0.06 Yes No T639R−0.48 0.10 −0.68 0.11 −0.16 0.35 No No T639N 0.03 0.16 −0.02 0.17 NA NAYes No T639D 0.61 0.17 0.43 0.18 NA NA Yes No T639E 0.71 0.12 0.82 0.12NA NA Yes No T639I 0.17 0.13 −0.19 0.14 NA NA Yes No T639L −0.28 0.15−0.39 0.16 NA NA Yes No T639P −0.10 0.12 0.08 0.12 −0.21 0.22 Yes NoT639S 0.07 0.07 0.09 0.07 −0.53 0.12 Yes No T639V 0.75 0.09 −1.09 0.140.78 0.28 Yes No K640A −0.31 0.07 −0.59 0.07 0.20 0.26 Yes No K640R−0.15 0.04 0.07 0.04 0.01 0.11 Yes No K640N 0.07 0.03 0.32 0.03 −0.430.05 Yes No K640D −0.14 0.09 −0.73 0.11 −0.52 0.30 No No K640C 0.06 0.080.34 0.08 −0.51 0.27 Yes No K640Q −0.86 0.12 0.36 0.09 0.29 0.31 Yes NoK640E −0.30 0.06 0.08 0.06 −0.03 0.15 Yes No K640G −0.26 0.04 −0.59 0.04−0.48 0.12 No No K640H 0.81 0.12 −0.26 0.15 NA NA Yes No K640I −0.100.12 −0.11 0.12 NA NA Yes No K640L 0.21 0.06 0.05 0.06 −0.31 0.20 Yes NoK640M −1.04 0.15 0.36 0.10 −0.12 0.31 Yes No K640F 0.24 0.12 −0.13 0.13NA NA Yes No K640P 0.40 0.09 0.14 0.10 −0.03 0.55 Yes No K640S −0.600.07 0.52 0.06 −0.11 0.22 Yes No K640* −0.85 0.10 −0.19 0.09 −0.96 0.21No No K640T 0.10 0.03 0.35 0.03 −0.52 0.06 Yes No K640W −0.53 0.07 0.410.06 0.12 0.25 Yes No K640Y −0.76 0.15 −0.28 0.14 0.36 0.37 Yes No K640V−0.26 0.05 −0.56 0.05 0.05 0.11 Yes No D840S 0.37 0.08 0.33 0.08 0.100.16 Yes Yes D840A −0.07 0.07 0.40 0.06 0.18 0.13 Yes No D840R −0.030.05 0.09 0.05 0.10 0.33 Yes No D840N −0.64 0.12 −0.31 0.11 −0.04 0.16No No D840C 0.62 0.12 −1.20 0.16 0.07 0.48 Yes No D840Q −0.22 0.14 −0.030.13 0.34 NA Yes No D840E 0.32 0.07 −0.07 0.07 0.08 0.06 Yes No D840G0.21 0.04 −0.26 0.04 0.02 0.05 Yes No D840L −0.64 0.10 −0.25 0.09 −0.270.82 No No D840K 0.13 0.13 0.33 0.12 −0.03 0.82 Yes No D840M NA NA NA NA−0.30 0.13 Yes No D840P −0.09 0.12 −0.12 0.12 0.11 0.82 Yes No D840T−1.14 0.14 0.11 0.10 0.29 0.15 Yes No D840W −0.33 0.09 0.05 0.08 −0.390.19 Yes No D840Y 0.13 0.08 0.11 0.08 −0.18 0.11 Yes No D840V 0.16 0.06−0.03 0.06 −0.10 0.07 Yes No E841A −0.26 0.06 −0.54 0.06 0.08 0.08 YesNo E841R −0.66 0.06 −0.57 0.05 −0.18 0.10 No No E841N 0.04 0.14 −0.530.15 −0.14 0.48 Yes No E841D −0.33 0.09 −0.04 0.08 0.12 0.15 Yes NoE841C −0.43 0.13 −0.39 0.12 −0.46 0.16 No No E841Q −0.64 0.12 −0.41 0.110.21 0.14 Yes No E841G 0.21 0.04 −0.11 0.04 −0.04 0.04 Yes No E841H−0.71 0.18 −0.82 0.18 NA NA Yes No E841L −1.39 0.10 −0.35 0.07 −0.410.06 No No E841K −0.55 0.09 −0.26 0.09 −0.34 0.17 No No E841M −1.21 0.14−0.41 0.11 −0.24 0.54 No No E841P −0.22 0.11 −0.23 0.11 0.00 0.08 No NoE841S −0.16 0.07 −0.22 0.07 0.04 0.10 Yes No E841* −0.19 0.06 −0.19 0.060.07 0.03 Yes No E841T 0.40 0.09 −0.59 0.10 −0.27 0.19 Yes No E841W−1.06 0.09 −1.15 0.09 −1.11 0.33 No No E841V −0.99 0.07 −0.68 0.06 −0.530.08 No No A842S 0.36 0.06 0.08 0.06 0.04 0.10 Yes Yes A842R −2.56 0.11−1.06 0.06 −1.25 0.15 No No A842D −0.37 0.11 −1.49 0.15 −1.07 0.19 No NoA842C 0.15 0.10 −0.39 0.11 −0.26 0.13 Yes No A842E NA NA NA NA −1.190.12 Yes No A842G −0.18 0.04 −0.18 0.04 −0.09 0.04 No No A842L −1.710.11 −1.29 0.09 −1.19 0.33 No No A842K −0.53 0.13 −1.41 0.16 NA NA YesNo A842P −0.87 0.10 −0.94 0.10 NA NA Yes No A842T −1.47 0.11 −0.57 0.08−1.03 0.10 No No A842W −1.87 0.12 −1.92 0.12 −1.46 0.40 No No A842V−1.40 0.08 −0.77 0.06 −1.17 0.20 No No R843A −0.28 0.10 0.30 0.09 0.150.11 Yes No R843E −1.03 0.17 −1.31 0.17 0.09 0.64 Yes No R843G 0.18 0.05−0.37 0.06 −0.43 0.07 Yes No R843L −0.31 0.13 −0.08 0.12 −0.05 0.30 NoNo R843K −0.16 0.13 0.01 0.12 0.45 0.16 Yes No R843M NA NA NA NA 0.260.83 Yes No R843S −0.08 0.07 0.08 0.07 −0.08 0.10 Yes No R843* −0.640.17 −0.23 0.15 NA NA Yes No R843T NA NA NA NA −0.19 0.30 Yes No R843W−0.99 0.15 0.22 0.12 −0.55 0.14 Yes No R843V −0.09 0.10 −0.23 0.10 0.040.17 Yes No A844E 0.38 0.10 0.06 0.10 0.13 0.21 Yes Yes A844R 0.45 0.07−0.04 0.07 0.03 0.37 Yes No A844D NA NA NA NA 0.36 0.11 Yes No A844C NANA NA NA 0.02 0.19 Yes No A844Q −0.26 0.18 0.84 0.15 −0.11 0.44 Yes NoA844G −0.48 0.06 −0.26 0.05 −0.01 0.06 No No A844L 0.54 0.10 −0.25 0.110.18 0.14 Yes No A844K NA NA NA NA 0.14 0.27 Yes No A844M NA NA NA NA0.23 0.41 Yes No A844P 0.28 0.11 −1.04 0.14 −0.72 0.25 Yes No A844S−0.22 0.08 −0.06 0.07 0.17 0.10 Yes No A844T −0.37 0.11 −0.05 0.10 0.220.21 Yes No A844W −1.01 0.13 −0.64 0.11 −0.10 NA No No A844V −0.19 0.080.12 0.07 0.06 0.08 Yes No L845A −0.70 0.08 −0.23 0.07 −0.59 0.11 No NoL845R −2.01 0.10 −1.83 0.09 −1.29 0.05 No No L845C −0.27 0.14 −0.84 0.15−0.37 0.82 No No L845Q NA NA NA NA −0.49 0.25 Yes No L845E −0.93 0.12−1.26 0.13 −0.79 0.48 No No L845G −1.31 0.06 −1.09 0.06 −1.46 0.20 No NoL845K −0.17 0.18 0.03 0.17 NA NA Yes No L845M −0.15 0.12 −0.17 0.12−0.20 0.25 No No L845P −0.64 0.10 −0.71 0.10 −0.61 0.11 No No L845S−1.48 0.13 −0.91 0.10 −0.82 0.35 No No L845T −0.85 0.14 0.43 0.11 NA NAYes No L845W −0.12 0.08 −1.89 0.13 −0.47 0.33 No No L845V −0.31 0.06−0.80 0.07 −0.20 0.10 No No L846A −1.10 0.08 −0.78 0.07 −0.78 0.82 No NoL846R −0.86 0.06 −0.85 0.05 −1.09 0.11 No No L846C −1.57 0.15 −1.32 0.13−0.93 0.14 No No L846Q −1.21 0.15 −0.87 0.13 −0.99 0.27 No No L846E−0.34 0.08 −0.41 0.08 −0.71 0.17 No No L846G −1.11 0.05 −0.81 0.05 −1.290.15 No No L846I NA NA NA NA 0.11 0.49 Yes No L846M −0.29 0.09 0.19 0.08−0.28 0.13 Yes No L846F −0.48 0.14 −0.27 0.13 NA NA Yes No L846P −1.040.10 −0.74 0.09 −0.74 0.13 No No L846S −0.77 0.09 −0.24 0.08 −1.23 0.37No No L846T −0.74 0.12 −1.37 0.14 −0.56 0.12 No No L846W −1.13 0.09−0.51 0.08 −0.35 0.06 No No L846Y −0.02 0.16 −0.98 0.19 NA NA Yes NoL846V −1.23 0.07 −0.61 0.06 −0.40 0.06 No No P847A 0.24 0.11 −1.48 0.16NA NA Yes No P847R −0.47 0.09 −0.88 0.10 −1.09 0.26 No No P847G −0.320.07 −1.12 0.08 −0.48 0.43 No No P847H −0.24 0.18 −0.19 0.17 −0.10 0.18No No P847L −1.09 0.12 −0.92 0.11 −0.76 0.15 No No P847S −0.42 0.11−0.55 0.11 −0.76 0.23 No No P847T −0.69 0.19 −0.89 0.19 NA NA Yes NoP847V −0.31 0.12 −1.08 0.14 NA NA Yes No N848A −0.53 0.11 0.02 0.10 0.140.43 Yes No N848R −0.30 0.07 −0.60 0.07 −0.35 0.10 No No N848D 0.06 0.11−0.08 0.11 −0.13 0.15 Yes No N848C −0.16 0.14 −0.15 0.14 −0.31 NA No NoN848Q −0.31 0.18 −0.55 0.19 NA NA Yes No N848E −0.64 0.11 0.35 0.09 0.480.09 Yes No N848G −0.87 0.07 −0.40 0.06 −0.33 0.11 No No N848H −0.070.05 0.05 0.05 −0.06 0.05 Yes No N848I 0.00 0.17 −0.09 0.17 NA NA Yes NoN848L 0.08 0.10 −1.50 0.14 −0.10 NA Yes No N848K −0.25 0.14 −0.45 0.14−0.20 0.19 No No N848M NA NA NA NA 0.10 0.64 Yes No N848S −0.89 0.10−0.37 0.08 −0.30 0.16 No No N848T 0.37 0.12 −0.11 0.12 −0.17 0.11 Yes NoN848W −0.76 0.12 −0.04 0.10 0.22 0.40 Yes No N848Y −0.61 0.18 0.16 0.16−0.20 0.24 Yes No N848V −0.51 0.09 −0.84 0.10 −0.29 0.64 No No V849A−0.93 0.12 0.41 0.09 −0.39 0.18 Yes No V849R −1.79 0.16 −1.96 0.16 −1.010.29 No No V849G −1.07 0.08 −1.40 0.08 −1.48 0.05 No No V849L 0.90 0.09−0.43 0.11 −0.12 0.13 Yes No V849M −0.12 0.12 −0.31 0.12 −0.19 0.19 NoNo I850A 0.25 0.05 −0.76 0.06 −0.17 0.06 Yes No I850R −0.32 0.04 −0.270.04 −0.21 0.06 No No I850N −0.38 0.13 −0.44 0.13 −0.50 0.10 No No I850D−1.19 0.12 −0.73 0.10 −0.71 0.54 No No I850C −0.30 0.08 0.19 0.08 0.130.14 Yes No I850Q −0.08 0.11 −0.31 0.11 0.00 0.43 No No I850E −0.24 0.07−0.45 0.07 −0.35 0.25 No No I850G −0.90 0.04 −0.86 0.04 −0.96 0.06 No NoI850H 0.45 0.12 −0.44 0.13 −0.29 0.54 Yes No I850L −0.15 0.04 −0.17 0.04−0.26 0.05 No No I850K −1.20 0.13 −0.19 0.10 −0.01 0.82 No No I850M−0.83 0.09 −0.82 0.09 −0.28 0.18 No No I850F −0.83 0.11 −0.97 0.11 −0.610.17 No No I850P −0.43 0.09 −1.72 0.12 −1.01 0.12 No No I850S −0.42 0.06−0.18 0.05 −0.52 0.06 No No I850T 0.00 0.07 −0.22 0.07 0.06 0.07 Yes NoI850W −1.02 0.07 −1.26 0.08 −0.97 0.10 No No I850V −0.03 0.04 0.12 0.040.22 0.05 Yes No T851A 0.41 0.06 0.14 0.06 0.08 0.10 Yes Yes T851V 0.320.06 0.13 0.06 0.54 0.07 Yes Yes T851R −0.18 0.06 −0.82 0.06 −0.93 0.17No No T851C 0.00 0.14 −0.03 0.14 −0.11 0.82 No No T851Q NA NA NA NA−0.17 0.29 Yes No T851E −0.44 0.09 −0.88 0.10 −0.08 0.08 No No T851G−1.22 0.06 −0.82 0.05 −0.95 0.19 No No T851I 0.21 0.13 −0.17 0.13 0.200.20 Yes No T851L 0.35 0.08 0.01 0.09 −0.05 0.37 Yes No T851K −0.23 0.14−0.49 0.14 NA NA Yes No T851M 0.52 0.11 −0.71 0.14 −0.02 0.23 Yes NoT851F −0.32 0.18 0.44 0.16 0.28 NA Yes No T851P −0.06 0.04 0.04 0.040.03 0.05 Yes No T851S −0.01 0.07 −0.04 0.07 −0.08 0.13 No No T851W 0.070.09 −0.34 0.09 −0.01 0.48 Yes No K852A −1.49 0.14 −1.41 0.13 NA NA YesNo K852R −0.07 0.06 −0.48 0.07 −0.37 0.10 No No K852N 0.12 0.10 −0.390.11 0.01 0.08 Yes No K852Q −0.04 0.05 0.10 0.04 0.14 0.06 Yes No K852E−0.99 0.12 −0.80 0.11 −1.03 0.07 No No K852G −1.42 0.09 −2.60 0.13 −1.200.18 No No K852P −0.25 0.07 −0.24 0.07 −0.22 0.08 No No K852S −1.14 0.13−0.56 0.10 −0.56 0.48 No No K852* −0.98 0.15 −1.24 0.16 NA NA Yes NoK852T −0.16 0.03 −0.02 0.03 0.16 0.04 Yes No K852V −0.65 0.09 −0.18 0.08−0.78 0.48 No No E853V 0.14 0.07 0.10 0.07 0.38 0.09 Yes Yes E853A −0.490.08 −0.35 0.08 −0.05 0.09 No No E853R 0.23 0.06 −0.13 0.06 −0.20 0.31Yes No E853D −0.30 0.10 −0.26 0.10 0.42 0.18 Yes No E853C 0.07 0.11 0.120.11 −0.24 0.13 Yes No E853Q −0.55 0.17 0.09 0.14 0.17 0.31 Yes No E853G−0.18 0.04 −0.41 0.05 −0.47 0.07 No No E853L −0.85 0.10 0.03 0.08 0.080.08 Yes No E853K 0.05 0.12 −0.95 0.15 0.21 0.20 Yes No E853M −0.08 0.15−0.94 0.17 0.01 0.11 Yes No E853P 0.46 0.13 −0.02 0.14 0.28 0.40 Yes NoE853S −0.30 0.09 −0.41 0.09 0.07 0.15 Yes No E853* −0.52 0.09 −0.38 0.09−0.25 0.08 No No E853T −0.33 0.15 −0.61 0.15 0.57 0.25 Yes No E853W 0.250.08 −1.68 0.12 −0.52 0.14 Yes No V854A −0.95 0.08 −0.31 0.06 −0.94 0.15No No V854R −1.42 0.07 −1.14 0.06 −1.31 0.13 No No V854D −0.58 0.13−1.22 0.15 NA NA Yes No V854C −0.91 0.13 −0.17 0.11 −0.54 0.35 No NoV854E −1.32 0.11 −1.01 0.09 −1.15 0.18 No No V854G −1.40 0.06 −1.44 0.06−1.28 0.09 No No V854L −1.49 0.10 −0.60 0.07 −1.28 0.13 No No V854M−1.32 0.13 −1.02 0.11 −0.94 0.16 No No V854P −1.29 0.16 −1.22 0.15 NA NAYes No V854S −1.58 0.12 −0.89 0.09 −1.26 0.21 No No V854W −1.48 0.10−0.37 0.07 −1.34 0.06 No No S855A 0.70 0.10 −0.77 0.12 0.01 0.40 Yes NoS855R 0.40 0.10 −0.66 0.11 0.61 0.12 Yes No S855C −0.24 0.17 0.37 0.15NA NA Yes No S855G 0.01 0.07 −0.69 0.08 −0.49 0.48 Yes No S855L NA NA NANA −0.20 0.82 Yes No S855F −0.02 0.17 0.31 0.16 NA NA Yes No S855P −0.310.12 0.14 0.10 −0.08 0.16 Yes No S855T 0.53 0.13 −0.42 0.15 0.41 0.24Yes No S855Y NA NA NA NA 0.21 0.29 Yes No S855V 0.17 0.13 0.00 0.14 0.240.82 Yes No H856R −0.04 0.07 −0.29 0.07 −0.62 0.16 No No H856N −1.010.17 −0.62 0.15 NA NA Yes No H856E −0.49 0.12 −0.23 0.11 NA NA Yes NoH856G −0.96 0.07 −1.35 0.07 −1.43 0.08 No No H856L −0.83 0.12 −0.29 0.10−0.42 0.20 No No H856P −0.26 0.09 0.07 0.09 −0.29 0.09 Yes No H856S−1.39 0.17 −0.13 0.12 NA NA Yes No H856W NA NA NA NA −0.62 0.17 Yes NoH856Y −0.65 0.16 −0.05 0.13 0.50 0.20 Yes No H856V NA NA NA NA −0.600.21 Yes No E857A −0.23 0.05 −0.30 0.05 −0.51 0.06 No No E857R −0.920.06 −1.30 0.06 −1.05 0.13 No No E857D 0.50 0.09 −0.31 0.10 −1.09 0.21Yes No E857C −0.44 0.13 −1.14 0.15 −0.59 0.40 No No E857Q −0.89 0.14−1.04 0.14 −0.73 0.29 No No E857G −0.98 0.04 −0.90 0.04 −1.19 0.03 No NoE857L −0.09 0.08 −0.75 0.09 −0.99 0.22 No No E857K 0.23 0.09 −0.93 0.11−0.76 0.14 Yes No E857M −0.36 0.13 −0.31 0.12 NA NA Yes No E857P −0.100.11 −0.53 0.12 −0.58 0.54 No No E857S −0.17 0.08 −0.67 0.08 −0.71 0.16No No E857* −1.28 0.15 −0.53 0.12 NA NA Yes No E857T −1.10 0.14 0.080.10 −0.09 0.54 Yes No E857W −0.75 0.08 −1.02 0.08 −1.03 0.15 No NoE857V −0.10 0.05 −0.43 0.05 −0.29 0.08 No No I858R −1.30 0.11 −1.37 0.10−1.32 0.24 No No I858G −2.03 0.11 −0.89 0.07 −1.33 0.09 No No I858L−0.32 0.08 −0.64 0.08 −0.25 0.08 No No I858F −0.53 0.17 −0.64 0.17 NA NAYes No I858S −1.55 0.16 −0.66 0.12 NA NA Yes No I858T −0.49 0.13 −0.720.13 −0.26 0.20 No No I858V −0.43 0.08 −0.43 0.08 −0.69 0.11 No No I859A−2.25 0.14 −1.44 0.10 −0.96 0.35 No No I859R −1.46 0.08 −1.27 0.07 −1.130.07 No No I859N −0.63 0.17 −0.59 0.16 −0.31 0.21 No No I859D −1.17 0.15−1.53 0.16 NA NA Yes No I859Q −0.35 0.17 0.41 0.15 NA NA Yes No I859E−0.25 0.09 −0.09 0.08 −0.48 0.11 No No I859G −1.65 0.07 −1.30 0.06 −1.320.22 No No I859L −0.20 0.04 −0.01 0.04 −0.26 0.04 No No I859M −0.66 0.120.38 0.10 −0.64 0.11 Yes No I859F −0.18 0.12 −0.15 0.12 −0.59 0.12 No NoI859S −0.34 0.08 −1.05 0.09 −1.12 0.17 No No I859T −0.26 0.09 −0.27 0.09−0.35 0.15 No No I859W −1.84 0.15 −0.31 0.09 −0.74 0.64 No No I859V 0.300.04 −0.13 0.05 0.24 0.05 Yes No K860A −0.50 0.06 −0.35 0.06 −1.06 0.07No No K860R −0.39 0.05 −0.83 0.05 −0.83 0.10 No No K860N −0.63 0.12−1.47 0.14 −0.88 0.16 No No K860C −0.36 0.10 −0.81 0.11 NA NA Yes NoK860Q −0.18 0.04 −0.19 0.04 −0.15 0.06 No No K860E −0.89 0.06 −1.15 0.07−1.08 0.05 No No K860G −1.23 0.05 −1.22 0.05 −1.28 0.06 No No K860L−0.68 0.07 −1.58 0.09 −1.19 0.11 No No K860M −0.24 0.09 −0.56 0.09 −1.000.15 No No K860P −0.21 0.07 −0.49 0.07 −0.50 0.09 No No K860S −1.38 0.08−1.80 0.09 −1.30 0.07 No No K860* −1.43 0.12 −1.23 0.11 −1.17 0.16 No NoK860T −0.15 0.04 −0.15 0.04 −0.07 0.06 No No K860W −1.18 0.08 −1.46 0.09−1.24 0.09 No No K860V −0.88 0.06 −1.87 0.08 −1.03 0.04 No No D861A−0.47 0.05 −0.76 0.06 −1.12 0.11 No No D861R −1.43 0.06 −1.83 0.06 −1.240.09 No No D861N −0.46 0.10 −0.59 0.10 −0.60 0.17 No No D861C −0.40 0.08−0.50 0.08 −0.69 0.08 No No D861Q −0.65 0.11 0.09 0.09 NA NA Yes NoD861E −0.97 0.07 −0.89 0.06 −0.50 0.12 No No D861G −0.85 0.03 −1.00 0.03−1.03 0.03 No No D861H 0.92 0.12 −0.60 0.15 −0.36 0.21 Yes No D861L−0.37 0.07 0.09 0.06 −0.18 0.27 Yes No D861K 0.55 0.09 −1.49 0.13 −1.220.43 Yes No D861M −0.29 0.10 −0.57 0.10 −0.68 0.40 No No D861F −1.050.15 −0.55 0.13 −0.22 0.12 No No D861S −1.43 0.09 −1.60 0.09 −1.45 0.28No No D861W −0.88 0.07 −0.84 0.07 −0.15 0.08 No No D861Y −0.18 0.17 0.140.16 −0.35 0.14 Yes No D861V −1.18 0.06 −0.99 0.05 −1.28 0.12 No NoR862K 0.04 0.14 0.54 0.13 0.02 0.38 Yes Yes R862A −1.76 0.13 −0.42 0.08−0.71 0.31 No No R862C −0.64 0.12 −1.78 0.16 −0.65 0.28 No No R862Q−0.27 0.10 −0.07 0.09 −0.28 0.13 No No R862E −0.64 0.12 −1.63 0.15 −0.760.09 No No R862G −0.72 0.05 −0.83 0.05 −0.85 0.07 No No R862L −0.90 0.08−0.16 0.07 −0.39 0.09 No No R862M −1.06 0.17 −0.69 0.15 NA NA Yes NoR862P −1.33 0.17 −1.10 0.15 NA NA Yes No R862W −1.35 0.11 −0.91 0.09−1.10 0.14 No No R862V NA NA NA NA −1.61 0.43 Yes No R863Q −0.49 0.13−0.79 0.14 NA NA Yes No R863G −1.68 0.09 −1.14 0.07 −1.45 0.07 No NoR863L −1.18 0.15 −1.68 0.17 −1.02 0.13 No No R863W −1.27 0.11 −1.06 0.10−1.17 0.14 No No F864I −0.25 0.11 −0.08 0.11 −0.19 0.12 No No F864L 0.010.07 −0.06 0.07 0.06 0.08 Yes No F864S −0.34 0.14 −0.27 0.14 −0.35 0.17No No F864W −0.66 0.18 −0.77 0.18 NA NA Yes No F864V −0.11 0.06 −0.130.05 0.00 0.08 No No T865A −0.65 0.14 −0.29 0.12 −0.09 0.18 No No T865N0.07 0.03 −0.05 0.03 −0.15 0.04 Yes No T865I −0.01 0.10 0.20 0.09 0.200.13 Yes No T865L NA NA NA NA 0.57 0.29 Yes No T865P −0.10 0.05 −0.070.05 −0.01 0.06 No No T865S −0.20 0.02 −0.20 0.02 0.01 0.03 Yes No T865Y−0.16 0.06 −0.17 0.05 −0.16 0.10 No No S866F 0.00 0.13 0.06 0.12 0.100.14 Yes Yes S866T 0.05 0.13 0.02 0.13 0.40 0.15 Yes Yes S866A −0.100.09 −0.40 0.10 0.05 0.07 Yes No S866R NA NA NA NA −0.33 0.09 Yes NoS866D NA NA NA NA 0.02 0.24 Yes No S866C NA NA NA NA −0.05 0.43 Yes NoS866Q NA NA NA NA −0.04 0.27 Yes No S866E NA NA NA NA 0.58 0.22 Yes NoS866G NA NA NA NA −0.97 0.29 Yes No S866L NA NA NA NA 0.57 0.10 Yes NoS866M NA NA NA NA 0.85 0.14 Yes No S866P 0.08 0.03 −0.07 0.03 0.00 0.03Yes No S866W NA NA NA NA −0.67 0.37 Yes No S866Y 0.13 0.16 −0.17 0.170.03 0.11 Yes No S866V NA NA NA NA 1.00 0.08 Yes No D867A 0.05 0.02−0.05 0.02 −0.05 0.02 Yes No D867R NA NA NA NA −1.39 0.06 Yes No D867NNA NA NA NA −0.17 0.14 Yes No D867C NA NA NA NA −0.98 0.17 Yes No D867E−0.16 0.11 −0.21 0.11 −0.99 0.09 No No D867G 0.06 0.02 0.00 0.02 −0.110.02 Yes No D867H −0.04 0.09 0.11 0.08 NA NA Yes No D867S NA NA NA NA−0.98 0.09 Yes No D867W NA NA NA NA −1.25 0.13 Yes No D867Y NA NA NA NA−1.02 0.12 Yes No D867V −0.04 0.10 −0.07 0.10 −0.92 0.07 No No K868A NANA NA NA −1.23 0.28 Yes No K868R −0.01 0.02 0.02 0.02 −0.76 0.04 Yes NoK868N NA NA NA NA −1.00 0.11 Yes No K868Q −0.14 0.09 −0.02 0.09 −0.110.08 No No K868E −0.43 0.19 −0.52 0.19 −1.33 0.19 No No K868G NA NA NANA −1.49 0.08 Yes No K868P NA NA NA NA −1.00 0.09 Yes No K868S NA NA NANA −1.32 0.15 Yes No K868* NA NA NA NA −1.02 0.18 Yes No K868T −0.150.03 −0.12 0.03 −0.14 0.07 No No K868W NA NA NA NA −1.50 0.15 Yes NoK868V NA NA NA NA −1.12 0.16 Yes No F869A NA NA NA NA −1.10 0.15 Yes NoF869R NA NA NA NA −1.47 0.27 Yes No F869C NA NA NA NA −1.20 0.15 Yes NoF869E NA NA NA NA −1.41 0.09 Yes No F869G NA NA NA NA −1.40 0.04 Yes NoF869I 0.06 0.10 −0.10 0.10 0.04 0.09 Yes No F869L −0.16 0.03 −0.17 0.03−0.01 0.06 No No F869M NA NA NA NA 0.14 0.09 Yes No F869S NA NA NA NA−1.16 0.09 Yes No F869W NA NA NA NA −1.32 0.12 Yes No F869Y NA NA NA NA−0.21 0.24 Yes No F869V −0.16 0.05 −0.18 0.05 −0.62 0.05 No No L870A−0.86 0.11 −0.83 0.10 NA NA Yes No L870R −1.55 0.08 −1.55 0.07 NA NA YesNo L870E −1.23 0.14 −1.60 0.15 NA NA Yes No L870G −1.54 0.07 −1.47 0.07NA NA Yes No L870M −0.94 0.16 −1.13 0.17 NA NA Yes No L870F −1.01 0.07−0.78 0.07 NA NA Yes No L870P −0.29 0.06 −0.36 0.06 NA NA Yes No L870V−0.67 0.08 −0.82 0.08 NA NA Yes No F870A NA NA NA NA −1.06 0.37 Yes NoF870R NA NA NA NA −1.61 0.14 Yes No F870C NA NA NA NA −0.66 0.43 Yes NoF870G NA NA NA NA −1.20 0.19 Yes No F870I NA NA NA NA 0.71 0.17 Yes NoF870L NA NA NA NA 1.11 0.06 Yes No F870M NA NA NA NA −0.04 0.44 Yes NoF870S NA NA NA NA −0.82 0.17 Yes No F870V NA NA NA NA 0.05 0.09 Yes NoF871R NA NA NA NA −1.55 0.28 Yes No F871C −0.24 0.17 −0.04 0.16 NA NAYes No F871G NA NA NA NA −1.08 0.22 Yes No F871L −0.17 0.07 −0.16 0.07−0.50 0.08 No No F871S NA NA NA NA −0.74 0.12 Yes No F871V 0.19 0.12−0.06 0.12 −0.68 0.22 Yes No H872R −0.49 0.19 −0.27 0.18 −1.33 0.16 NoNo H872Q −0.40 0.10 −0.32 0.10 −0.60 0.11 No No H872G NA NA NA NA −1.610.11 Yes No H872L 0.00 0.08 −0.15 0.08 −0.43 0.08 No No H872P 0.04 0.02−0.01 0.02 0.34 0.02 Yes No H872Y NA NA NA NA 0.10 0.21 Yes No H872V NANA NA NA −1.51 0.48 Yes No V873A 0.16 0.11 0.05 0.11 0.08 0.16 Yes YesV873E −0.40 0.14 −0.35 0.14 NA NA Yes No V873G −0.13 0.04 −0.20 0.03−0.27 0.07 No No V873L −0.20 0.05 0.24 0.05 −0.40 0.08 Yes No V873M−0.48 0.12 −0.33 0.11 −0.16 0.16 No No P874A −0.12 0.06 0.06 0.06 0.030.11 Yes No P874G NA NA NA NA −0.22 0.17 Yes No P874S −0.06 0.10 −0.170.10 0.58 0.11 Yes No P874T −0.23 0.06 −0.24 0.06 −0.09 0.04 No No I875R−0.33 0.12 −0.24 0.11 −0.82 0.10 No No I875N −0.01 0.07 −0.09 0.07 0.030.08 Yes No I875G NA NA NA NA −1.64 0.10 Yes No I875H 0.10 0.11 −0.010.11 −0.25 0.11 Yes No I875L 0.08 0.02 0.02 0.02 −0.08 0.02 Yes No I875M−0.44 0.17 −0.26 0.16 0.05 0.17 Yes No I875F −0.20 0.09 −0.13 0.09 −0.120.10 No No I875S −0.06 0.07 −0.04 0.07 −0.22 0.07 No No I875T −0.01 0.10−0.10 0.09 −0.11 0.11 No No I875V −0.24 0.17 −0.16 0.16 −0.44 0.10 No NoT876A NA NA NA NA −1.21 0.20 Yes No T876R NA NA NA NA −1.72 0.26 Yes NoT876N −0.10 0.03 −0.09 0.03 −0.07 0.05 No No T876G NA NA NA NA −1.440.22 Yes No T876P −0.03 0.06 −0.07 0.06 −0.30 0.05 No No T876S NA NA NANA −0.63 0.19 Yes No T876V NA NA NA NA −1.34 0.33 Yes No L877A 0.09 0.12−0.51 0.13 NA NA Yes No L877R −0.89 0.08 −1.39 0.09 −1.53 0.08 No NoL877C −0.26 0.17 −0.47 0.17 NA NA Yes No L877Q −0.43 0.11 −0.45 0.11−0.39 0.13 No No L877G −1.42 0.09 −2.38 0.13 −1.74 0.35 No No L877P−0.08 0.07 −0.22 0.07 −0.40 0.12 No No L877S −1.11 0.16 −0.99 0.15 NA NAYes No L877V −1.25 0.14 0.04 0.10 −0.96 0.08 Yes No L877A −0.03 0.10−0.76 0.11 NA NA Yes No L877R −0.15 0.04 −0.31 0.04 −0.02 0.04 No NoL877C −0.29 0.15 −0.65 0.15 NA NA Yes No L877Q −0.26 0.11 −0.47 0.11 NANA Yes No L877E NA NA NA NA −0.74 0.64 Yes No L877G −1.40 0.08 −2.820.12 −1.79 0.22 No No L877M −0.19 0.11 0.18 0.10 0.26 0.10 Yes No L877P−0.28 0.09 −0.39 0.08 −0.46 0.15 No No L877S −1.31 0.14 −1.01 0.12 NA NAYes No L877V −1.10 0.10 −0.29 0.08 −0.58 0.06 No No N878D 0.03 0.06 0.020.06 0.04 0.08 Yes Yes N878A 0.17 0.16 −0.41 0.17 −0.06 0.25 Yes NoN878Q −0.22 0.17 −0.53 0.17 NA NA Yes No N878E −0.73 0.15 −0.44 0.13−0.84 0.13 No No N878G −0.57 0.10 −0.98 0.11 −0.70 0.06 No No N878H−0.01 0.05 −0.10 0.05 0.03 0.05 Yes No N878L NA NA NA NA −0.13 0.64 YesNo N878K −0.01 0.02 −0.05 0.02 −0.06 0.02 No No N878S −1.08 0.15 −0.430.12 −0.38 0.10 No No N878T −0.16 0.06 0.09 0.06 0.15 0.07 Yes No N878Y−0.05 0.07 0.00 0.06 0.06 0.04 Yes No N878V −1.01 0.17 −0.32 0.14 −0.190.34 No No N878A 0.03 0.14 −0.32 0.14 −0.19 0.54 Yes No N878D −0.11 0.12−0.34 0.12 −0.62 0.15 No No N878C NA NA NA NA 0.58 0.42 Yes No N878G−0.62 0.10 −1.09 0.11 −0.81 0.05 No No N878I −0.35 0.16 −0.02 0.15 −0.410.17 No No N878L NA NA NA NA −0.12 0.82 Yes No N878K −0.34 0.13 −0.400.13 −0.39 0.08 No No N878M NA NA NA NA 0.25 0.16 Yes No N878S −0.540.11 −0.30 0.10 −0.50 0.07 No No N878T −0.19 0.05 −0.20 0.05 −0.37 0.09No No N878Y −0.34 0.18 −0.28 0.17 −0.12 0.21 No No N878V −0.98 0.15−0.37 0.12 −0.51 0.32 No No Y879A −2.04 0.15 −1.14 0.10 NA NA Yes NoY879R −0.78 0.08 −1.40 0.08 −1.48 0.11 No No Y879D 0.39 0.05 −0.19 0.06−0.18 0.07 Yes No Y879C 0.50 0.06 −0.02 0.07 −0.50 0.09 Yes No Y879Q−0.72 0.19 −0.38 0.17 NA NA Yes No Y879E −1.40 0.13 −1.23 0.11 NA NA YesNo Y879G −0.58 0.05 −1.93 0.07 −1.84 0.23 No No Y879H 0.35 0.09 0.040.09 −0.16 0.12 Yes No Y879L −1.45 0.13 0.26 0.08 −1.39 0.33 Yes NoY879K 0.13 0.14 −1.11 0.17 NA NA Yes No Y879F −0.05 0.12 −0.01 0.11 0.010.17 Yes No Y879S 0.37 0.04 −0.29 0.04 −0.43 0.07 Yes No Y879* −0.200.11 0.92 0.09 −0.81 0.15 Yes No Y879W −0.05 0.08 −0.07 0.07 0.60 0.07Yes No Y879V −0.53 0.08 −0.97 0.08 NA NA Yes No Q880R 0.13 0.05 0.020.05 0.01 0.06 Yes Yes Q880G 0.25 0.04 0.19 0.04 0.64 0.05 Yes Yes Q880A−0.10 0.08 0.40 0.07 −0.19 0.20 Yes No Q880D 0.81 0.14 −1.10 0.18 NA NAYes No Q880C −0.08 0.14 −1.19 0.17 0.17 0.14 Yes No Q880E −0.73 0.10−1.06 0.10 −0.58 0.08 No No Q880H 0.24 0.08 −0.08 0.08 −0.10 0.08 Yes NoQ880L −0.80 0.08 −0.59 0.07 −1.39 0.07 No No Q880K −0.23 0.13 −0.27 0.13−0.44 0.09 No No Q880M −0.93 0.16 −0.77 0.14 −0.49 0.17 No No Q880P−0.07 0.10 −0.40 0.10 0.12 0.07 Yes No Q880S −0.31 0.09 −0.76 0.10 −0.090.07 No No Q880* −1.27 0.14 −0.76 0.11 −1.05 0.11 No No Q880T −0.52 0.14−0.18 0.12 0.07 0.29 Yes No Q880W −0.68 0.08 −0.91 0.08 −0.82 0.06 No NoQ880V −1.77 0.10 −0.81 0.07 −0.63 0.07 No No A881R −0.95 0.07 −1.75 0.08−1.43 0.18 No No A881D −0.36 0.11 −0.32 0.10 −0.48 0.19 No No A881C−0.35 0.14 −0.78 0.15 −0.49 0.10 No No A881G 0.14 0.04 0.32 0.04 −0.410.04 Yes No A881L −1.36 0.11 −2.39 0.15 NA NA Yes No A881P −1.05 0.16−0.99 0.14 −0.80 0.18 No No A881S 0.13 0.05 0.13 0.05 −0.13 0.06 Yes NoA881T −0.73 0.10 −0.95 0.10 −0.44 0.14 No No A881W −1.25 0.12 −1.48 0.12NA NA Yes No A881V −1.66 0.09 −1.28 0.07 −0.91 0.08 No No A882D 0.320.05 0.22 0.05 0.10 0.07 Yes Yes A882S 0.11 0.12 0.14 0.11 0.23 0.22 YesYes A882R −0.10 0.10 0.54 0.09 0.51 0.35 Yes No A882G −0.43 0.07 −0.320.06 −0.14 0.06 No No A882L −1.02 0.19 0.32 0.14 NA NA Yes No A882P−0.12 0.14 −0.11 0.13 −0.12 0.24 No No A882T −0.14 0.09 0.05 0.09 −0.090.13 Yes No A882V 0.39 0.08 0.04 0.08 −0.17 0.15 Yes No N883G 0.42 0.080.37 0.08 0.03 0.11 Yes Yes N883K 0.12 0.16 0.23 0.16 0.16 0.10 Yes YesN883A −0.26 0.15 −1.10 0.17 0.05 0.55 Yes No N883R NA NA NA NA 0.84 0.11Yes No N883D −0.02 0.13 −0.37 0.14 −0.36 0.18 No No N883I 0.00 0.16 0.080.15 −0.37 0.12 Yes No N883S 0.08 0.10 0.04 0.10 −0.13 0.09 Yes No N883T0.27 0.11 −0.02 0.11 −0.21 0.12 Yes No S884V 0.05 0.11 0.61 0.10 0.030.14 Yes Yes S884A 0.07 0.13 −0.01 0.12 0.02 NA Yes No S884R −0.28 0.06−0.11 0.06 0.34 0.07 Yes No S884N 0.14 0.14 0.48 0.13 −0.07 0.25 Yes NoS884C NA NA NA NA 0.18 0.19 Yes No S884E −0.10 0.16 −0.04 0.15 NA NA YesNo S884G −0.09 0.06 −0.11 0.06 0.01 0.10 Yes No S884L NA NA NA NA 0.020.55 Yes No S884T −0.01 0.10 0.05 0.10 NA NA Yes No S884W −0.99 0.18−0.38 0.14 0.43 0.82 Yes No P885A −0.36 0.06 −0.32 0.05 −0.12 0.13 No NoP885R −0.03 0.04 −0.89 0.05 0.00 0.06 Yes No P885N −0.47 0.17 −0.73 0.17NA NA Yes No P885D −0.73 0.12 −1.21 0.12 −0.83 0.35 No No P885C −1.540.14 −0.39 0.09 −0.01 0.09 No No P885Q −1.26 0.18 −0.04 0.13 −0.65 0.33No No P885E −0.08 0.08 −1.01 0.09 −0.98 0.16 No No P885G −1.22 0.05−0.98 0.04 −0.39 0.04 No No P885H −0.06 0.13 −0.41 0.13 0.09 0.12 Yes NoP885I NA NA NA NA −0.01 0.44 Yes No P885L −0.66 0.07 −0.90 0.07 −0.600.05 No No P885K −0.12 0.12 −0.28 0.12 −0.32 0.22 No No P885M −1.32 0.15−1.01 0.13 −0.52 0.13 No No P885F −0.98 0.16 −0.49 0.14 −0.31 0.10 No NoP885S −0.88 0.07 −0.65 0.06 −0.11 0.12 No No P885T −0.35 0.04 −0.29 0.040.03 0.05 Yes No P885W −0.76 0.07 −1.20 0.08 −0.94 0.23 No No P885Y−0.19 0.16 0.72 0.13 0.22 0.48 Yes No P885V −0.86 0.06 −0.46 0.05 0.050.12 Yes No S886L 0.09 0.08 0.12 0.08 0.05 0.11 Yes Yes S886M 0.32 0.110.15 0.11 0.40 0.35 Yes Yes S886W 0.23 0.07 0.01 0.07 0.70 0.19 Yes YesS886A −0.52 0.07 −0.42 0.07 −0.04 0.14 No No S886R 0.12 0.04 −0.08 0.040.36 0.04 Yes No S886N 0.34 0.10 −0.34 0.11 −0.03 0.14 Yes No S886D 0.600.10 −0.69 0.12 NA NA Yes No S886C 0.11 0.09 −0.64 0.09 0.34 0.23 Yes NoS886Q −0.33 0.14 −0.65 0.14 0.19 0.64 Yes No S886E −0.60 0.10 −0.56 0.10NA NA Yes No S886G −0.20 0.04 −0.18 0.04 0.10 0.05 Yes No S886I 0.890.13 −0.13 0.14 0.07 0.29 Yes No S886K −0.70 0.14 0.01 0.12 0.56 0.12Yes No S886F NA NA NA NA 0.75 0.44 Yes No S886P NA NA NA NA −0.28 0.14Yes No S886T 0.07 0.07 −0.05 0.07 −0.29 0.10 Yes No S886Y NA NA NA NA0.79 0.55 Yes No S886V −0.10 0.07 −0.15 0.06 0.35 0.20 Yes No K887A 0.030.04 −0.38 0.05 −0.54 0.04 Yes No K887R −0.09 0.03 −0.04 0.03 0.21 0.03Yes No K887N 0.24 0.10 0.07 0.10 −0.45 0.24 Yes No K887D −1.19 0.10−0.97 0.09 −1.27 0.28 No No K887C −0.76 0.08 0.16 0.07 −0.59 0.06 Yes NoK887Q 0.10 0.08 −0.05 0.07 −0.25 0.11 Yes No K887E −0.33 0.05 −0.39 0.05−0.97 0.06 No No K887G −0.14 0.03 −0.22 0.02 −0.42 0.03 No No K887H−0.04 0.12 −1.21 0.15 −0.21 0.40 No No K887I −0.31 0.12 −0.04 0.11 NA NAYes No K887L −0.88 0.06 −0.67 0.05 −0.68 0.07 No No K887M −0.12 0.07−0.23 0.07 −0.44 0.16 No No K887F 0.22 0.11 −0.22 0.11 −0.74 0.25 Yes NoK887P −0.29 0.08 −0.61 0.08 −0.32 0.18 No No K887S −0.03 0.05 −0.50 0.05−0.66 0.08 No No K887* −0.88 0.08 −1.30 0.08 −1.22 0.10 No No K887T−0.79 0.08 −0.22 0.07 −0.74 0.11 No No K887W −0.59 0.05 −0.69 0.05 −0.650.07 No No K887Y 0.36 0.10 −0.98 0.13 −0.40 0.17 Yes No K887V 0.09 0.04−0.88 0.04 −0.89 0.07 Yes No F888A −2.01 0.10 −1.31 0.07 −1.30 0.28 NoNo F888R −1.27 0.07 −1.04 0.06 −1.82 0.18 No No F888D −1.09 0.12 −1.320.12 NA NA Yes No F888C 0.01 0.04 −0.10 0.04 0.10 0.03 Yes No F888Q−0.58 0.13 −1.37 0.15 NA NA Yes No F888E −0.89 0.08 −2.09 0.11 −1.770.12 No No F888G −1.75 0.05 −1.53 0.05 −1.57 0.03 No No F888I 0.09 0.11−0.24 0.11 −0.50 0.15 Yes No F888L −0.13 0.04 −0.06 0.03 −0.23 0.05 NoNo F888M −0.65 0.12 0.31 0.10 −0.04 0.10 Yes No F888P −0.69 0.12 −1.560.15 NA NA Yes No F888S −1.42 0.08 −1.17 0.07 −1.14 0.12 No No F888*−1.06 0.13 −0.77 0.11 −1.31 0.08 No No F888T −0.62 0.11 −1.69 0.13 −1.190.64 No No F888W −1.15 0.08 −2.14 0.11 −1.37 0.05 No No F888Y −0.73 0.13−1.27 0.14 −0.60 0.12 No No F888V 0.07 0.04 0.10 0.04 −0.31 0.03 Yes NoN889A NA NA NA NA −0.86 0.64 Yes No N889R −0.94 0.09 −0.07 0.07 −0.550.48 No No N889D −0.34 0.10 −0.44 0.10 −0.39 0.14 No No N889G −0.87 0.06−1.32 0.07 −0.83 0.07 No No N889I −0.51 0.17 −0.43 0.16 NA NA Yes NoN889K −0.76 0.17 −0.47 0.15 −0.34 0.24 No No N889S −1.13 0.11 0.08 0.08−0.26 0.14 Yes No N889T −0.55 0.16 −0.45 0.14 −0.36 0.30 No No N889V−1.82 0.15 −1.28 0.12 −1.16 0.64 No No Q890C 0.03 0.11 0.64 0.10 0.080.54 Yes Yes Q890H 0.39 0.09 0.07 0.09 0.04 0.11 Yes Yes Q890W 0.03 0.080.23 0.07 0.03 0.08 Yes Yes Q890A 0.39 0.06 0.05 0.06 −0.02 0.11 Yes NoQ890R −0.10 0.05 −0.24 0.05 −0.05 0.10 No No Q890N −0.25 0.15 −0.66 0.160.31 NA Yes No Q890D 0.54 0.10 −0.52 0.11 −0.35 0.20 Yes No Q890E 0.170.06 −0.62 0.07 −0.33 0.06 Yes No Q890G −0.35 0.04 −0.60 0.04 −0.15 0.07No No Q890I −0.26 0.16 −0.94 0.18 NA NA Yes No Q890L 0.09 0.06 −0.140.06 0.29 0.17 Yes No Q890K 0.23 0.10 0.25 0.10 −0.22 0.16 Yes No Q890M0.32 0.11 −0.13 0.11 −0.07 0.48 Yes No Q890F 0.25 0.15 −0.11 0.15 0.090.82 Yes No Q890P −1.20 0.12 −0.75 0.10 −0.11 0.17 No No Q890S −0.580.08 −0.01 0.07 −0.19 0.08 No No Q890* −1.12 0.11 −1.26 0.11 −0.88 0.13No No Q890T −0.36 0.10 0.11 0.09 0.08 0.09 Yes No Q890Y NA NA NA NA 0.030.13 Yes No Q890V −0.39 0.06 −0.33 0.06 0.21 0.06 Yes No R891A −0.410.08 −0.46 0.07 −0.44 0.31 No No R891D −1.17 0.16 −0.76 0.14 −0.63 0.64No No R891C −0.10 0.10 −0.56 0.11 −0.05 0.17 No No R891Q NA NA NA NA−0.08 0.64 Yes No R891E −1.06 0.11 −0.65 0.09 −0.59 0.64 No No R891G−0.70 0.04 −0.40 0.04 −0.11 0.04 No No R891L 0.07 0.08 −0.02 0.08 −0.250.33 Yes No R891K −0.04 0.11 −0.37 0.11 −0.03 0.26 No No R891M NA NA NANA −0.14 NA Yes No R891S −0.18 0.07 −0.49 0.07 −0.28 0.08 No No R891*−0.88 0.13 −0.57 0.11 −0.74 0.20 No No R891T −0.20 0.10 −0.28 0.10 −0.160.10 No No R891W −1.03 0.08 −1.42 0.08 −0.78 0.43 No No R891V −0.28 0.07−0.77 0.07 −0.53 0.09 No No V892A −1.02 0.08 −1.08 0.08 −0.18 0.07 No NoV892R −1.49 0.09 −1.55 0.08 −1.41 0.09 No No V892C −0.58 0.14 −0.80 0.14−0.01 0.13 No No V892E −0.97 0.10 −1.28 0.10 −0.93 0.17 No No V892G−1.01 0.05 −1.11 0.05 −0.90 0.05 No No V892L −0.58 0.09 −1.35 0.10 −0.930.14 No No V892M −0.59 0.12 −0.62 0.12 NA NA Yes No V892S −1.15 0.13−1.21 0.12 NA NA Yes No V892T −0.34 0.15 0.05 0.13 0.11 0.64 Yes NoV892W −1.99 0.15 −1.09 0.10 NA NA Yes No N893A −1.02 0.07 −0.18 0.05−0.37 0.05 No No N893R −0.35 0.04 −0.57 0.04 −0.42 0.06 No No N893D−0.71 0.10 −0.39 0.08 −0.71 0.11 No No N893C −0.80 0.10 −0.20 0.09 −0.240.07 No No N893Q −1.58 0.15 −0.86 0.11 −0.57 0.20 No No N893E −0.89 0.07−1.03 0.07 −1.10 0.10 No No N893G −0.30 0.03 −0.68 0.03 −0.34 0.04 No NoN893H 0.02 0.13 −0.74 0.14 NA NA Yes No N893I 0.33 0.11 −0.24 0.12 −0.680.11 Yes No N893L −1.03 0.08 −0.46 0.07 −0.86 0.17 No No N893K −0.390.07 −0.07 0.06 −0.27 0.07 No No N893M 0.26 0.08 −0.81 0.10 −1.04 0.33Yes No N893S 0.64 0.05 0.08 0.05 −0.47 0.06 Yes No N893* −1.54 0.12−1.43 0.11 −1.26 0.17 No No N893T 1.09 0.06 −0.04 0.06 −0.62 0.08 Yes NoN893W −0.63 0.06 −0.48 0.06 −0.78 0.10 No No N893Y −0.51 0.12 −0.87 0.12−0.38 0.18 No No N893V −0.70 0.06 −0.74 0.06 −0.59 0.05 No No A894R 0.190.06 0.06 0.06 0.04 0.08 Yes Yes A894D 0.04 0.06 0.09 0.06 0.21 0.05 YesYes A894C −0.13 0.15 −0.29 0.14 NA NA Yes No A894Q 0.87 0.14 −0.47 0.16NA NA Yes No A894E −0.35 0.10 −0.85 0.10 −0.45 0.20 No No A894G −0.470.05 0.09 0.05 0.21 0.06 Yes No A894L −0.45 0.11 0.57 0.09 0.08 0.12 YesNo A894K NA NA NA NA 0.27 0.15 Yes No A894M 0.14 0.13 −0.17 0.13 0.250.18 Yes No A894P −0.67 0.14 −1.05 0.15 NA NA Yes No A894S 0.57 0.08−0.97 0.10 0.12 0.23 Yes No A894* −0.76 0.19 0.06 0.16 NA NA Yes NoA894T −0.68 0.10 −0.49 0.09 0.21 0.15 Yes No A894W 0.67 0.09 −0.44 0.100.02 0.54 Yes No A894V −0.22 0.07 −0.05 0.06 0.12 0.18 Yes No Y895D 0.160.04 0.00 0.04 0.09 0.04 Yes Yes Y895A −1.08 0.12 −0.85 0.11 −0.53 0.37No No Y895R NA NA NA NA −0.75 0.07 Yes No Y895N −0.12 0.11 −0.43 0.11−0.07 0.23 No No Y895C 0.61 0.06 0.14 0.06 −0.26 0.07 Yes No Y895E −0.530.11 −0.74 0.10 −0.62 0.13 No No Y895G −0.59 0.06 −1.69 0.08 −1.72 0.07No No Y895H −0.21 0.10 −0.10 0.10 −0.25 0.16 No No Y895F 0.70 0.12 −0.280.13 −0.20 0.21 Yes No Y895S 0.18 0.05 −0.17 0.05 −0.12 0.06 Yes NoY895* −0.03 0.05 −0.04 0.05 0.16 0.06 Yes No Y895W −0.08 0.11 0.17 0.10−0.18 0.31 Yes No Y895V −1.60 0.12 −0.11 0.08 −0.61 0.40 No No L896A−1.48 0.09 −1.26 0.08 −1.68 0.35 No No L896R 0.19 0.04 −0.11 0.04 −0.300.04 Yes No L896Q 0.42 0.04 0.08 0.04 −0.17 0.05 Yes No L896E −1.31 0.11−1.65 0.12 NA NA Yes No L896G −2.17 0.07 −1.66 0.05 −1.77 0.05 No NoL896M 0.31 0.11 −0.22 0.11 −0.74 0.17 Yes No L896F −0.69 0.18 −0.97 0.18NA NA Yes No L896P −0.37 0.08 −0.52 0.08 −0.53 0.10 No No L896S NA NA NANA −1.38 0.48 Yes No L896W −1.69 0.12 −1.64 0.11 −0.97 0.54 No No L896V−0.04 0.06 −0.69 0.06 −0.81 0.07 No No K897A −0.76 0.08 −0.22 0.07 −0.070.15 No No K897R 0.03 0.04 −0.01 0.04 −0.05 0.05 Yes No K897N 0.58 0.110.26 0.11 −0.06 0.09 Yes No K897D −1.39 0.13 −0.61 0.10 −0.10 0.25 No NoK897C 0.02 0.09 −0.54 0.10 −0.11 0.08 Yes No K897Q −0.06 0.10 0.16 0.10−0.04 0.21 Yes No K897E 0.17 0.05 −0.03 0.05 −0.29 0.08 Yes No K897G0.01 0.04 −0.29 0.04 −0.07 0.03 Yes No K897H −0.18 0.15 0.06 0.14 0.220.36 Yes No K897I −0.29 0.13 −1.02 0.14 −0.06 0.06 No No K897L −0.190.07 −0.26 0.07 0.28 0.35 Yes No K897M −0.35 0.09 −0.01 0.08 0.23 0.11Yes No K897F −0.72 0.18 0.16 0.15 −0.08 0.64 Yes No K897P −1.22 0.13−1.78 0.14 NA NA Yes No K897S −0.22 0.07 −0.29 0.07 −0.13 0.10 No NoK897* −0.92 0.11 −0.93 0.11 −0.93 0.11 No No K897T 0.27 0.08 −0.32 0.08−0.02 0.06 Yes No K897W −0.08 0.07 −0.54 0.07 0.00 0.19 No No K897Y−0.95 0.19 0.19 0.14 −0.23 0.13 Yes No K897V −0.30 0.05 −0.29 0.05 0.190.07 Yes No E898A −0.19 0.06 0.08 0.06 −0.25 0.07 Yes No E898R −0.440.07 −0.49 0.07 −0.10 0.31 No No E898D 0.42 0.10 −0.32 0.11 −0.12 0.16Yes No E898C NA NA NA NA 0.23 0.15 Yes No E898Q −0.43 0.14 −0.57 0.130.29 0.39 Yes No E898G −0.27 0.05 −0.27 0.05 0.12 0.06 Yes No E898L 0.070.11 −0.46 0.12 0.15 0.12 Yes No E898K −0.93 0.16 −0.35 0.14 0.13 0.31Yes No E898P −0.40 0.19 −0.60 0.19 NA NA Yes No E898S 0.17 0.14 −1.320.18 0.38 0.55 Yes No E898W −1.01 0.12 −1.33 0.12 −0.16 0.14 No No E898V−0.06 0.07 0.31 0.07 0.01 0.07 Yes No H899A 0.21 0.08 0.16 0.07 0.010.48 Yes Yes H899N 0.15 0.15 0.70 0.14 0.08 0.18 Yes Yes H899R −0.200.04 −0.12 0.04 −0.12 0.03 No No H899D −0.84 0.13 −0.16 0.11 −0.47 0.25No No H899C −0.98 0.14 −0.02 0.11 −0.12 0.13 No No H899Q −0.15 0.05−0.20 0.05 0.00 0.03 No No H899E −0.85 0.10 −0.64 0.09 −0.36 0.27 No NoH899G −0.79 0.05 −0.96 0.05 −0.65 0.05 No No H899L −0.27 0.08 −0.41 0.08−0.50 0.14 No No H899K NA NA NA NA 0.05 0.49 Yes No H899P −0.07 0.070.01 0.07 −0.33 0.10 Yes No H899S −0.31 0.10 −0.33 0.09 0.07 0.32 Yes NoH899T −0.37 0.15 −0.14 0.14 −0.03 0.48 No No H899W −0.32 0.08 −0.24 0.07−0.25 0.48 No No H899Y −0.09 0.12 −0.87 0.13 0.11 0.16 Yes No H899V−0.07 0.07 −0.99 0.08 0.05 0.22 Yes No P900A −0.09 0.05 0.03 0.04 0.040.11 Yes No P900R −0.17 0.04 −0.23 0.03 0.09 0.04 Yes No P900N −1.130.14 0.05 0.11 0.35 0.30 Yes No P900D −0.42 0.08 −0.19 0.07 0.19 0.12Yes No P900C −0.28 0.07 0.15 0.06 0.12 0.15 Yes No P900Q 0.28 0.08 −0.190.09 0.23 0.30 Yes No P900E −0.19 0.06 0.18 0.05 0.29 0.08 Yes No P900G−0.03 0.03 −0.15 0.03 0.11 0.03 Yes No P900H 0.07 0.08 −0.18 0.08 0.250.12 Yes No P900I −0.83 0.12 −0.36 0.10 0.33 0.33 Yes No P900L 0.03 0.04−0.20 0.04 0.07 0.04 Yes No P900K 0.16 0.08 −0.61 0.08 0.05 0.13 Yes NoP900M −1.26 0.10 −0.21 0.07 0.06 0.12 Yes No P900F 0.33 0.10 −0.07 0.10−0.19 0.09 Yes No P900S −0.10 0.05 0.15 0.04 0.14 0.04 Yes No P900*−0.32 0.08 −1.30 0.10 NA NA Yes No P900T 0.24 0.06 −0.22 0.06 0.19 0.09Yes No P900W −0.16 0.05 −0.48 0.06 −0.04 0.06 No No P900Y −0.49 0.11−0.86 0.11 −0.25 0.82 No No P900V 0.10 0.04 −0.35 0.04 0.14 0.05 Yes NoE901D 0.07 0.10 0.05 0.10 0.14 0.15 Yes Yes E901A −0.81 0.08 0.24 0.070.08 0.08 Yes No E901R −0.15 0.06 −0.19 0.06 −0.03 0.06 No No E901C−0.08 0.13 −0.32 0.13 0.21 0.64 Yes No E901Q −0.69 0.16 0.04 0.13 −0.160.40 Yes No E901G −0.44 0.04 0.07 0.04 0.10 0.06 Yes No E901L −0.12 0.08−0.80 0.09 −0.13 0.20 No No E901K −0.11 0.10 0.20 0.10 0.08 0.13 Yes NoE901M 0.16 0.12 −0.14 0.12 0.03 0.82 Yes No E901P −0.99 0.19 −1.23 0.19NA NA Yes No E901S −1.40 0.13 0.16 0.08 0.05 0.11 Yes No E901* −0.290.13 −1.11 0.15 −0.63 0.14 No No E901T NA NA NA NA −0.05 0.44 Yes NoE901W 0.02 0.08 −0.49 0.09 0.21 0.08 Yes No E901V −1.13 0.07 −0.49 0.060.11 0.06 Yes No T902A −0.07 0.05 −0.35 0.05 −0.20 0.09 No No T902R−0.60 0.06 −1.09 0.07 −0.48 0.12 No No T902C 0.17 0.11 −0.55 0.12 −0.250.82 Yes No T902Q −0.92 0.15 −0.24 0.13 NA NA Yes No T902E −0.55 0.10−1.12 0.11 NA NA Yes No T902G −1.03 0.06 −1.00 0.06 −0.74 0.15 No NoT902I −0.22 0.14 0.19 0.12 −0.10 0.20 Yes No T902L −0.92 0.11 −0.09 0.09−0.12 0.09 No No T902K −0.31 0.04 −0.01 0.04 0.13 0.06 Yes No T902M NANA NA NA 0.01 0.54 Yes No T902P −1.06 0.13 −0.32 0.10 −0.81 0.21 No NoT902S −0.15 0.07 −0.38 0.07 −0.15 0.07 No No T902W NA NA NA NA −0.800.54 Yes No T902V −0.15 0.07 0.06 0.07 −0.03 0.14 Yes No P903T 0.64 0.130.52 0.12 0.39 0.18 Yes Yes P903A −0.22 0.11 −0.37 0.10 −0.12 0.31 No NoP903R −1.86 0.13 −0.87 0.09 −0.31 0.27 No No P903C 0.75 0.15 −0.71 0.18NA NA Yes No P903E NA NA NA NA 0.05 0.22 Yes No P903G −1.37 0.10 −1.660.10 −0.49 0.35 No No P903H NA NA NA NA 0.04 0.17 Yes No P903L −0.520.11 −0.93 0.12 0.29 0.10 Yes No P903S −0.61 0.12 0.03 0.10 0.07 0.22Yes No P903V −0.44 0.12 −0.77 0.12 0.25 0.16 Yes No I904A 0.28 0.05 0.320.05 −0.01 0.10 Yes No I904R −0.52 0.05 −0.28 0.04 −0.14 0.04 No NoI904N −0.26 0.11 0.07 0.10 −0.29 0.13 Yes No I904D NA NA NA NA −1.330.37 Yes No I904C −0.37 0.09 −0.06 0.08 0.16 0.09 Yes No I904Q −0.280.11 0.17 0.10 0.03 0.37 Yes No I904E −1.15 0.08 −1.73 0.09 −0.77 0.30No No I904G −1.05 0.04 −0.45 0.04 −0.39 0.03 No No I904H NA NA NA NA−0.20 0.48 Yes No I904L −0.35 0.07 0.47 0.06 0.12 0.09 Yes No I904K−1.71 0.15 −0.48 0.10 0.05 0.09 Yes No I904M 0.19 0.08 −0.04 0.08 0.030.05 Yes No I904F −0.71 0.13 0.38 0.11 0.15 0.20 Yes No I904S 0.20 0.04−0.09 0.04 0.07 0.03 Yes No I904* −1.37 0.12 −1.64 0.13 NA NA Yes NoI904T 0.33 0.07 −0.20 0.07 −0.13 0.08 Yes No I904W −0.01 0.06 −0.07 0.06−0.35 0.24 No No I904Y 0.53 0.12 −0.47 0.13 0.01 0.64 Yes No I904V −0.130.05 0.08 0.04 0.00 0.04 Yes No I905A −0.31 0.06 −0.49 0.06 −0.02 0.14No No I905R −1.93 0.08 −1.23 0.06 −1.47 0.09 No No I905N −0.95 0.15−0.68 0.14 NA NA Yes No I905D −1.69 0.14 −1.47 0.12 NA NA Yes No I905C−0.01 0.10 0.49 0.09 −0.12 0.11 Yes No I905E −1.48 0.10 −2.09 0.12 −1.570.43 No No I905G −1.25 0.05 −0.83 0.04 −0.80 0.04 No No I905L −0.65 0.07−0.31 0.06 −0.12 0.07 No No I905M 0.15 0.06 −0.40 0.07 −0.10 0.06 Yes NoI905F −0.10 0.11 −0.09 0.11 −0.55 0.18 No No I905S −1.01 0.07 −1.06 0.07−0.63 0.06 No No I905T −0.17 0.09 −0.86 0.09 −0.52 0.12 No No I905W−1.31 0.10 −1.78 0.11 −1.43 0.28 No No I905V −0.18 0.05 −0.21 0.05 0.100.06 Yes No G906A 0.41 0.05 0.56 0.04 0.42 0.05 Yes Yes G906S 0.40 0.050.16 0.05 0.15 0.06 Yes Yes G906R −1.82 0.06 −2.39 0.07 −1.63 0.08 No NoG906D −1.49 0.10 −1.05 0.08 −1.38 0.18 No No G906C −1.50 0.11 −1.21 0.09−1.62 0.11 No No G906E −2.26 0.12 −1.63 0.09 −1.49 0.11 No No G906L−1.51 0.09 −2.32 0.11 −1.69 0.27 No No G906P −1.68 0.12 −1.05 0.09 −1.660.10 No No G906* NA NA NA NA −1.21 0.40 Yes No G906T −1.69 0.11 −2.680.15 NA NA Yes No G906W −2.22 0.11 −1.14 0.07 −1.71 0.08 No No G906V−0.85 0.05 −1.20 0.05 −1.27 0.06 No No I907A 0.70 0.10 0.42 0.10 0.270.54 Yes Yes I907V 0.15 0.07 0.12 0.07 0.13 0.06 Yes Yes I907R −0.920.08 −1.15 0.08 −0.89 0.09 No No I907D −0.44 0.17 −0.97 0.18 NA NA YesNo I907C −0.16 0.16 0.84 0.13 0.12 0.64 Yes No I907E −1.06 0.15 −0.420.12 NA NA Yes No I907G −0.42 0.06 −0.40 0.06 −0.03 0.06 No No I907L0.34 0.09 0.24 0.08 −0.33 0.09 Yes No I907M NA NA NA NA 0.12 0.25 Yes NoI907F −0.50 0.19 −0.17 0.17 NA NA Yes No I907S −0.29 0.03 −0.18 0.030.25 0.03 Yes No I907T −0.44 0.15 0.78 0.12 0.28 0.12 Yes No D908A −2.260.13 −1.92 0.11 −1.10 0.16 No No D908R NA NA NA NA −1.53 0.09 Yes NoD908N −0.27 0.12 −0.30 0.12 −0.70 0.14 No No D908E −1.61 0.13 −1.42 0.11−1.11 0.11 No No D908G −2.25 0.09 −2.15 0.08 −1.77 0.14 No No D908S−1.92 0.16 −1.78 0.14 NA NA Yes No D908W −1.93 0.15 −2.11 0.15 NA NA YesNo D908Y 0.26 0.11 −0.07 0.12 −0.15 0.12 Yes No D908V −1.74 0.10 −1.760.10 −1.52 0.09 No No R909A NA NA NA NA −1.45 0.37 Yes No R909Q −0.250.18 −0.94 0.20 NA NA Yes No R909G −1.30 0.05 −1.49 0.05 −1.38 0.06 NoNo R909K −0.87 0.16 −0.60 0.14 NA NA Yes No R909S −1.08 0.10 −0.84 0.09−0.58 0.12 No No R909T −1.28 0.15 −0.89 0.13 NA NA Yes No R909W −0.960.10 −1.94 0.13 NA NA Yes No R909V −1.36 0.11 −2.33 0.14 −1.34 0.47 NoNo G910R −1.03 0.08 −1.62 0.09 −1.75 0.16 No No G910D NA NA NA NA −0.690.19 Yes No G910C −0.91 0.13 −1.29 0.14 NA NA Yes No G910S −1.15 0.10−1.28 0.10 −1.41 0.19 No No G910W −1.93 0.15 −1.82 0.13 NA NA Yes NoG910V −1.34 0.08 −1.15 0.07 −0.66 0.13 No No E911A −0.79 0.12 −1.56 0.14−0.93 0.10 No No E911R −1.42 0.12 −2.19 0.14 NA NA Yes No E911D −0.450.14 −0.47 0.13 −0.18 0.25 No No E911G −0.65 0.06 −0.80 0.06 −0.83 0.08No No E911L NA NA NA NA −0.50 0.15 Yes No E911K −0.39 0.13 −0.14 0.12−0.57 0.15 No No E911* −0.20 0.11 0.12 0.10 −0.18 0.16 Yes No E911V−0.21 0.08 −0.62 0.08 −0.61 0.09 No No R912A 0.46 0.09 0.49 0.09 0.310.64 Yes Yes R912L 0.53 0.08 0.38 0.08 0.21 0.16 Yes Yes R912S 1.04 0.110.30 0.11 0.31 0.54 Yes Yes R912V 0.19 0.09 0.23 0.09 0.03 0.09 Yes YesR912C 0.43 0.15 0.54 0.15 −0.29 0.16 Yes No R912Q 0.22 0.05 0.15 0.05−0.05 0.09 Yes No R912E 0.53 0.12 −0.67 0.14 NA NA Yes No R912G −0.210.05 −1.04 0.06 −0.56 0.05 No No R912P 0.53 0.09 0.79 0.09 −0.40 0.14Yes No R912T −0.54 0.17 −0.07 0.15 0.52 0.65 Yes No R912W −0.09 0.07−0.14 0.07 −0.43 0.10 No No N913A −1.19 0.13 −0.97 0.11 −0.87 0.33 No NoN913R −2.49 0.14 −0.64 0.07 −1.67 0.40 No No N913D −0.62 0.13 −0.44 0.12−0.75 0.10 No No N913E −0.91 0.12 −1.92 0.16 NA NA Yes No N913G −1.340.07 −0.91 0.06 −1.56 0.30 No No N913H 0.12 0.07 0.13 0.07 −0.20 0.12Yes No N913I −0.16 0.09 −0.18 0.09 −0.18 0.12 No No N913L −0.39 0.11−0.72 0.11 NA NA Yes No N913K −0.37 0.05 −0.29 0.04 −0.27 0.08 No NoN913S −0.29 0.08 −0.06 0.07 −0.20 0.12 No No N913T 0.09 0.11 −0.67 0.12−0.35 0.24 Yes No N913Y −0.47 0.18 −0.44 0.17 NA NA Yes No N913V −1.320.12 −0.89 0.10 −1.09 0.18 No No ¹Key to Header abbreviations: Mutantsare defined by single letter amino acid (wild-type) at a given positionin the wild-type AsCas12a polypeptide (e.g., amino acid position 500),followed by the variant amino acid change thereafter; “Enrich. Score(1)” refers to Enrichment score of the variant in a Condition 1background; “Stand. Error (1) refers to the Standard error forexperiments conducted for a given variant in a Condition 1 background;“Enrich. Score (2)” refers to Enrichment Score of the variant in aCondition 2 background; “Stand. Error (2) refers to the Standard Errorfor experiments conducted for a given variant in a Condition 2background; “Enrich. Score (3)”refers to Enrichment Score of the variantin a Condition 3 background; “Stand. Error (3) refers to the StandardError for experiments conducted for a given variant in a Condition 3background; “Any Positive Enrichment?” refers to the occurrence ofpositive enrichment for a given variant under at least one experimentalCondition background; “Consistent positive enrichment?” refers to theoccurrence of positive enrichment for a given variant under all testedexperimental Condition backgrounds.

One hundred eighty-seven (187) variants (˜6% of total) consistentlyenhanced the survival rate under all conditions (Table 4). Thesevariants, including the four validated individually, can be stacked onthe WT- or M537R/F870L-AsCas12a, to boost its DNA cleavage activity atTTTT-PAM.

TABLE 4 Point mutations consistently enriched over the referenceproteins under all conditions.¹ SEQ ID Variant score Variant scoreVariant score NO.: Mutant Position (condition 1) (condition 2)(condition 3)  59 R499C 499 0.14 ± 0.08  0.6 ± 0.07 0.06 ± 0.1   60R499L 499 0.08 ± 0.05 0.05 ± 0.06 0.09 ± 0.15  61 R499K 499 0.54 ± 0.060.23 ± 0.06 0.38 ± 0.1   62 L500M 500 0.01 ± 0.05  0.3 ± 0.05 0.28 ±0.12  63 T501L 501 0.18 ± 0.04 0.32 ± 0.04 0.12 ± 0.07  64 T501M 5010.32 ± 0.07 0.35 ± 0.07 0.17 ± 0.29  65 T501V 501 0.22 ± 0.06 0.29 ±0.06 0.13 ± 0.2   66 G502R 502 0.14 ± 0.05 0.28 ± 0.05 0.98 ± 0.07  67G502E 502 0.42 ± 0.08 0.17 ± 0.08 0.19 ± 0.3   68 G502L 502 0.45 ± 0.07 0.1 ± 0.07   0 ± 0.11  69 G502S 502 0.08 ± 0.06 0.23 ± 0.06 0.16 ± 0.12 70 G502W 502 0.14 ± 0.07 0.65 ± 0.06 0.36 ± 0.14  71 G502V 502 0.21 ±0.05 0.21 ± 0.05 0.13 ± 0.07  72 L505A 505 0.61 ± 0.05  0.5 ± 0.05 0.47± 0.13  73 L505R 505  0.4 ± 0.03 0.56 ± 0.03 0.81 ± 0.05  74 L505Q 505 0.3 ± 0.07 0.82 ± 0.07 0.16 ± 0.11  75 L505E 505 0.18 ± 0.06 0.03 ±0.07 0.14 ± 0.21  76 L505G 505 0.18 ± 0.03 0.72 ± 0.03  0.4 ± 0.05  77L505H 505 0.33 ± 0.11 0.83 ± 0.1  0.34 ± 0.32  78 L505K 505 0.02 ± 0.1   1 ± 0.08   1 ± 0.13  79 L505S 505 0.13 ± 0.06 0.32 ± 0.06  0.6 ± 0.07 80 E506A 506 0.33 ± 0.03 0.47 ± 0.03  0.7 ± 0.05  81 E506R 506 0.46 ±0.03 0.81 ± 0.03  1.2 ± 0.04  82 E506N 506 0.24 ± 0.09 0.53 ± 0.09  0.5± 0.29  83 E506C 506  0.2 ± 0.06 0.29 ± 0.06 0.49 ± 0.19  84 E506Q 5060.09 ± 0.07 0.74 ± 0.06 0.33 ± 0.15  85 E506G 506 0.24 ± 0.02 0.39 ±0.02 0.53 ± 0.03  86 E506H 506 0.35 ± 0.09 0.1 ± 0.1 0.98 ± 0.31  87E506I 506 0.47 ± 0.09 0.45 ± 0.09 0.69 ± 0.13  88 E506L 506  0.3 ± 0.040.61 ± 0.04 0.56 ± 0.07  89 E506K 506 0.18 ± 0.06 0.59 ± 0.05 1.08 ±0.08  90 E506M 506 0.53 ± 0.06 0.28 ± 0.07 0.47 ± 0.17  91 E506S 5060.01 ± 0.05 0.47 ± 0.04  0.5 ± 0.06  92 E506T 506 0.39 ± 0.06 0.12 ±0.07  0.9 ± 0.23  93 E506Y 506 0.16 ± 0.09 0.29 ± 0.09 0.43 ± 0.25  94E506V 506 0.45 ± 0.03 0.51 ± 0.03 0.75 ± 0.05  95 E508A 508 0.36 ± 0.03 0.2 ± 0.03 0.18 ± 0.06  96 E508R 508 0.54 ± 0.03  0.8 ± 0.03 0.82 ±0.06  97 E508Q 508 0.25 ± 0.06 0.11 ± 0.07 0.51 ± 0.13  98 E508G 5080.16 ± 0.02 0.17 ± 0.02 0.22 ± 0.04  99 E508L 508 0.03 ± 0.04  0.1 ±0.04 0.26 ± 0.11 100 E508K 508  0.2 ± 0.06 0.49 ± 0.06 0.66 ± 0.08 101E508M 508 0.25 ± 0.07 0.57 ± 0.06 0.54 ± 0.11 102 E508F 508 0.27 ± 0.080.19 ± 0.08 0.39 ± 0.29 103 E508S 508 0.31 ± 0.05 0.62 ± 0.04 0.34 ±0.06 104 E508T 508 0.19 ± 0.06 0.73 ± 0.06 0.55 ± 0.1  105 E508Y 5080.35 ± 0.09 0.19 ± 0.1  0.21 ± 0.27 106 E508V 508 0.16 ± 0.03 0.22 ±0.03 0.34 ± 0.05 107 P509R 509 0.23 ± 0.03 0.27 ± 0.03 1.04 ± 0.05 108P509K 509 0.01 ± 0.07 0.12 ± 0.07 1.29 ± 0.13 109 P509M 509 0.34 ± 0.070.34 ± 0.07  0.1 ± 0.22 110 P509S 509  0.1 ± 0.04 0.14 ± 0.04 0.14 ±0.1  111 P509W 509 0.04 ± 0.05 0.33 ± 0.05 0.61 ± 0.18 112 P509Y 5090.18 ± 0.09  0.2 ± 0.09 0.53 ± 0.3  113 S510G 510 0.18 ± 0.03 0.49 ±0.03 0.21 ± 0.06 114 S510L 510 0.57 ± 0.06 0.72 ± 0.06 0.74 ± 0.09 115S512R 512 0.07 ± 0.07 0.34 ± 0.07 0.07 ± 0.19 116 F513L 513 0.53 ± 0.040.63 ± 0.04 0.41 ± 0.07 117 F513W 513 0.04 ± 0.06 0.35 ± 0.06 0.28 ±0.13 118 N515A 515  0.6 ± 0.06 0.82 ± 0.06 0.12 ± 0.11 119 N515R 5150.63 ± 0.06 0.82 ± 0.05 0.19 ± 0.11 120 N515I 515 0.23 ± 0.07  0.3 ±0.07 0.16 ± 0.14 121 N515L 515 0.33 ± 0.06 0.53 ± 0.06 0.07 ± 0.2  122N515T 515 0.41 ± 0.08 0.14 ± 0.08 0.29 ± 0.16 123 N515V 515  0.5 ± 0.050.49 ± 0.05 0.45 ± 0.09 124 K516R 516 0.01 ± 0.03 0.26 ± 0.03  0.4 ±0.06 125 R518K 518  0.2 ± 0.09 0.72 ± 0.08 0.05 ± 0.17 126 T522L 522 1.1 ± 0.05 0.92 ± 0.05 0.28 ± 0.1  127 T522M 522 0.92 ± 0.07 0.81 ±0.08 0.53 ± 0.21 128 T522V 522 0.16 ± 0.05 0.28 ± 0.05 0.04 ± 0.07 129K523R 523 0.17 ± 0.02 0.37 ± 0.02   0 ± 0.03 130 S527D 527 0.13 ± 0.110.01 ± 0.11 0.34 ± 0.41 131 V528D 528 0.01 ± 0.07 0.27 ± 0.07 0.12 ±0.12 132 V528L 528 0.55 ± 0.03 0.59 ± 0.03 0.31 ± 0.07 133 V528M 5280.19 ± 0.05 0.02 ± 0.05 0.13 ± 0.1  134 S542N 542 0.24 ± 0.09 0.02 ±0.09 0.29 ± 0.07 135 S542C 542 0.03 ± 0.17 0.15 ± 0.17 0.42 ± 0.16 136K550R 550 0.05 ± 0.03 0.18 ± 0.03 0.17 ± 0.07 137 N551D 551 0.14 ± 0.040.15 ± 0.04 0.67 ± 0.07 138 A554I 554 0.15 ± 0.09 0.71 ± 0.08 0.39 ±0.34 139 A554T 554 0.45 ± 0.04 0.32 ± 0.05 0.93 ± 0.07 140 A554V 5540.57 ± 0.03 0.48 ± 0.03 0.54 ± 0.04 141 I555V 555 0.41 ± 0.03 0.13 ±0.03 0.14 ± 0.06 142 L556M 556 0.08 ± 0.08  0.1 ± 0.08 0.11 ± 0.21 143V558M 558 0.24 ± 0.06 0.15 ± 0.06  0.1 ± 0.12 144 K559A 559 0.17 ± 0.040.08 ± 0.04 0.13 ± 0.08 145 N560A 560 0.34 ± 0.05 0.18 ± 0.05 0.18 ±0.08 146 N560D 560 0.16 ± 0.04 0.18 ± 0.04 0.16 ± 0.08 147 N560E 5600.48 ± 0.06 0.28 ± 0.06 0.11 ± 0.25 148 N560M 560 0.16 ± 0.09 0.31 ±0.09 0.18 ± 0.13 149 P569D 569  0.6 ± 0.08 0.33 ± 0.08 1.25 ± 0.11 150Q571A 571 0.64 ± 0.07 0.39 ± 0.08 1.09 ± 0.23 151 Q571P 571 0.17 ± 0.070.61 ± 0.07 0.13 ± 0.14 152 Q571S 571 0.25 ± 0.09  0.2 ± 0.09 0.89 ±0.23 153 Q571T 571 0.22 ± 0.13 0.19 ± 0.13 0.87 ± 0.41 154 K572E 5720.11 ± 0.04 0.25 ± 0.04 0.42 ± 0.08 155 Y575G 575 0.53 ± 0.04 0.21 ±0.04 0.32 ± 0.07 156 Y575M 575 0.89 ± 0.11 0.27 ± 0.12 0.31 ± 0.41 157K576C 576 0.27 ± 0.07 0.25 ± 0.07  0.1 ± 0.26 158 S579T 579 0.17 ± 0.040.33 ± 0.04 0.01 ± 0.06 159 S579V 579 0.04 ± 0.03 0.02 ± 0.03 0.05 ±0.07 160 T583I 583 0.04 ± 0.08 0.11 ± 0.07 0.07 ± 0.17 161 E584H 5840.09 ± 0.07 0.22 ± 0.07 0.17 ± 0.14 162 E584V 584  0.1 ± 0.03 0.06 ±0.03 0.12 ± 0.04 163 K585R 585 0.07 ± 0.03 0.32 ± 0.03 0.06 ± 0.05 164K585F 585 0.51 ± 0.09 0.04 ± 0.09 0.61 ± 0.36 165 D596E 596 0.04 ± 0.040.24 ± 0.04 0.69 ± 0.05 166 P599G 599 0.84 ± 0.04 0.83 ± 0.04 1.01 ±0.05 167 A602C 602 0.64 ± 0.09 0.61 ± 0.09 0.73 ± 0.28 168 L612M 612 0.7 ± 0.07  0.7 ± 0.07 0.01 ± 0.03 169 A614R 614 0.28 ± 0.04 0.07 ±0.05 0.16 ± 0.05 170 A614I 614 0.52 ± 0.11 0.59 ± 0.11 0.41 ± 0.22 171T616A 616 0.32 ± 0.03 0.24 ± 0.04 0.09 ± 0.05 172 T616R 616 0.19 ± 0.030.12 ± 0.03 0.62 ± 0.04 173 T616Q 616 0.35 ± 0.07 0.38 ± 0.07 0.78 ±0.2  174 T616G 616 0.22 ± 0.03 0.01 ± 0.03 0.16 ± 0.06 175 T616Y 6160.54 ± 0.09 0.64 ± 0.09 0.05 ± 0.38 176 A617G 617 0.03 ± 0.03 0.05 ±0.03 0.24 ± 0.06 177 F619M 619 0.12 ± 0.07 0.55 ± 0.07 0.22 ± 0.25 178Q620A 620 0.22 ± 0.04   0 ± 0.05 0.18 ± 0.09 179 Q620R 620 0.25 ± 0.030.18 ± 0.03 0.17 ± 0.04 180 Q620N 620 0.26 ± 0.14 0.28 ± 0.14 0.36 ±0.21 181 Q620L 620 0.14 ± 0.04 0.45 ± 0.04  0.4 ± 0.16 182 Q620K 620 0.1 ± 0.07 0.24 ± 0.07 0.04 ± 0.1  183 T621A 621 0.18 ± 0.04 0.13 ±0.04 0.17 ± 0.1  184 H622G 622 0.05 ± 0.03 0.18 ± 0.03 0.07 ± 0.06 185H622S 622 0.16 ± 0.06 0.59 ± 0.06 0.08 ± 0.1  186 H622T 622 0.01 ± 0.090.84 ± 0.08 0.31 ± 0.34 187 H622V 622 0.18 ± 0.05 0.15 ± 0.05 0.31 ±0.12 188 T623E 623 0.32 ± 0.05 0.23 ± 0.05 0.02 ± 0.05 189 T623H 623 0.6± 0.1 0.01 ± 0.11 0.58 ± 0.55 190 T623L 623 0.33 ± 0.05 0.18 ± 0.05 0.17± 0.16 191 T623M 623 0.21 ± 0.07 0.28 ± 0.07 0.59 ± 0.22 192 T623F 6230.8 ± 0.1 0.61 ± 0.1  0.47 ± 0.32 193 T624P 624 0.92 ± 0.02 0.92 ± 0.020.04 ± 0.03 194 L627C 627 0.14 ± 0.13 0.84 ± 0.11 0.57 ± 0.48 195 L628C628 0.31 ± 0.09 0.1 ± 0.1 0.29 ± 0.31 196 L628W 628 0.25 ± 0.06 0.01 ±0.07 0.67 ± 0.15 197 N630R 630 0.53 ± 0.05 0.06 ± 0.06 0.94 ± 0.08 198I633N 633 0.16 ± 0.06 0.65 ± 0.06 0.15 ± 0.1  199 I633M 633 0.17 ± 0.070.28 ± 0.07  0.1 ± 0.19 200 I633S 633 0.02 ± 0.04 0.35 ± 0.04 0.22 ±0.08 201 E634N 634 0.52 ± 0.12 0.46 ± 0.12   0 ± 0.36 202 P635A 635 0.04± 0.05 0.29 ± 0.05 0.59 ± 0.07 203 P635D 635 0.14 ± 0.09 0.49 ± 0.090.52 ± 0.14 204 P635E 635 0.51 ± 0.06 0.49 ± 0.06  0.7 ± 0.16 205 P635T635 0.36 ± 0.06 0.06 ± 0.06 0.01 ± 0.08 206 T639G 639 0.52 ± 0.06 0.44 ±0.06 0.06 ± 0.23 207 D840S 840 0.37 ± 0.08 0.33 ± 0.08  0.1 ± 0.16 208A842S 842 0.36 ± 0.06 0.08 ± 0.06 0.04 ± 0.1  209 A844E 844 0.38 ± 0.1 0.06 ± 0. 1 0.13 ± 0.21 210 T851A 851 0.41 ± 0.06 0.14 ± 0.06 0.08 ±0.1  211 T851V 851 0.32 ± 0.06 0.13 ± 0.06 0.54 ± 0.07 212 E853V 8530.14 ± 0.07  0.1 ± 0.07 0.38 ± 0.09 213 R862K 862 0.04 ± 0.14 0.54 ±0.13 0.02 ± 0.38 214 S866F 866   0 ± 0.13 0.06 ± 0.12  0.1 ± 0.14 215S866T 866 0.05 ± 0.13 0.02 ± 0.13  0.4 ± 0.15 216 V873A 873 0.16 ± 0.110.05 ± 0.11 0.08 ± 0.16 217 N878D 878 0.03 ± 0.06 0.02 ± 0.06 0.04 ±0.08 218 Q880R 880 0.13 ± 0.05 0.02 ± 0.05 0.01 ± 0.06 219 Q880G 8800.25 ± 0.04 0.19 ± 0.04 0.64 ± 0.05 220 A882D 882 0.32 ± 0.05 0.22 ±0.05  0.1 ± 0.07 221 A882S 882 0.11 ± 0.12 0.14 ± 0.11 0.23 ± 0.22 222N883G 883 0.42 ± 0.08 0.37 ± 0.08 0.03 ± 0.11 223 N883K 883 0.12 ± 0.160.23 ± 0.16 0.16 ± 0.1  224 S884V 884 0.05 ± 0.11 0.61 ± 0.1  0.03 ±0.14 225 S886L 886 0.09 ± 0.08 0.12 ± 0.08 0.05 ± 0.11 226 S886M 8860.32 ± 0.11 0.15 ± 0.11  0.4 ± 0.35 227 S886W 886 0.23 ± 0.07 0.01 ±0.07  0.7 ± 0.19 228 Q890C 890 0.03 ± 0.11 0.64 ± 0.1  0.08 ± 0.54 229Q890H 890 0.39 ± 0.09 0.07 ± 0.09 0.04 ± 0.11 230 Q890W 890 0.03 ± 0.080.23 ± 0.07 0.03 ± 0.08 231 A894R 894 0.19 ± 0.06 0.06 ± 0.06 0.04 ±0.08 232 A894D 894 0.04 ± 0.06 0.09 ± 0.06 0.21 ± 0.05 233 Y895D 8950.16 ± 0.04   0 ± 0.04 0.09 ± 0.04 234 H899A 899 0.21 ± 0.08 0.16 ± 0.070.01 ± 0.48 235 H899N 899 0.15 ± 0.15  0.7 ± 0.14 0.08 ± 0.18 236 E901D901 0.07 ± 0.1  0.05 ± 0.1  0.14 ± 0.15 237 P903T 903 0.64 ± 0.13 0.52 ±0.12 0.39 ± 0.18 328 G906A 906 0.41 ± 0.05 0.56 ± 0.04 0.42 ± 0.05 239G906S 906  0.4 ± 0.05 0.16 ± 0.05 0.15 ± 0.06 240 1907A 907 0.7 ± 0.10.42 ± 0.1  0.27 ± 0.54 241 1907V 907 0.15 ± 0.07 0.12 ± 0.07 0.13 ±0.06 242 R912A 912 0.46 ± 0.09 0.49 ± 0.09 0.31 ± 0.64 243 R912L 9120.53 ± 0.08 0.38 ± 0.08 0.21 ± 0.16 244 R912S 912 1.04 ± 0.11  0.3 ±0.11 0.31 ± 0.54 245 R912V 912 0.19 ± 0.09 0.23 ± 0.09 0.03 ± 0.09 ¹Thephenotype scores (i.e. natural logarithm of relative enrichment) of eachpoint mutation are provided. The error bar estimates the precision ofthe measurement, which is dependent on the sequencing count of eachvariant in the libraries. Only variants with counts greater than 50 inall libraries were included in the analysis.

Table 5 shows the sequences used as primers to generate the AsCas12asaturation mutagenesis library. Standard recombinant methods andtechniques were used. The screening library was constructed using themethod described in Wrenbeck et al. (2016).

TABLE 5 Primer sequences to generate AsCas12a saturation mutagenesislibrary. SEQ ID NO.: Name Sequence (5′-3′)^(a) 246 499_NNK_HsCas12a_R499gaccccgagttctccgccNNKctgacaggcatcaaactg 247 500_NNK_HsCas12a_L500ggaccccgagttctccgccagaNNKacaggcatcaaac 248 501_NNK_HsCas12a_T501cgagttctccgccagactgNNKggcatcaaactggaaatgg 249 502_NNK_HsCas12a_G502gagttctccgccagactgacaNNKatcaaactggaaatggaaccc 250 503_NNK_HsCas12a_I503ctccgccagactgacaggcNNKaaactggaaatggaaccca 251 504_NNK_HsCas12a_K504gccagactgacaggcatcNNKctggaaatggaacccagc 252 505_NNK_HsCas12a_L505ccagactgacaggcatcaaaNNKgaaatggaacccagcctgtc 253 506_NNK_HsCas12a_E506gactgacaggcatcaaactgNNKatggaacccagcctgtcctt 254 507_NNK_HsCas12a_M507ctgacaggcatcaaactggaaNNKgaacccagcctgtccttctac 255 508_NNK_HsCas12a_E508ctgacaggcatcaaactggaaatgNNKcccagcctgtccttc 256 509_NNK_HsCas12a_P509gactgacaggcatcaaactggaaatggaaNNKagcctgtccttctac 257510_NNK_HsCas12a_S510 atcaaactggaaatggaacccNNKctgtccttctacaacaaggcc 258511_NNK_HsCas12a_L511 aaactggaaatggaacccagcNNKtccttctacaacaaggccaga 259512_NNK_HsCas12a_S512 ctggaaatggaacccagcctgNNKttctacaacaaggccagaaac 260513_NNK_HsCas12a_F513 gaaatggaacccagcctgtccNNKtacaacaaggccagaaactac 261514_NNK_HsCas12a_Y514 ggaacccagcctgtccttcNNKaacaaggccagaaactacg 262515_NNK_HsCas12a_N515 cccagcctgtccttctacNNKaaggccagaaactacgcc 263516_NNK_HsCas12a_K516 cagcctgtccttctacaacNNKgccagaaactacgccacca 264517_NNK_HsCas12a_A517 cagcctgtccttctacaacaagNNKagaaactacgccaccaagaaac265 518_NNK_HsCas12a_R518 ctgtccttctacaacaaggccNNKaactacgccaccaagaaaccc266 519_NNK_HsCas12a_N519 tccttctacaacaaggccagaNNKtacgccaccaagaaaccctac267 520_NNK_HsCas12a_Y520 ttctacaacaaggccagaaacNNKgccaccaagaaaccctacagc268 521_NNK_HsCas12a_A521 tacaacaaggccagaaactacNNKaccaagaaaccctacagcgtg269 522_NNK_HsCas12a_T522 caaggccagaaactacgccNNKaagaaaccctacagcgtgg 270523_NNK_HsCas12a_K523 ggccagaaactacgccaccNNKaaaccctacagcgtggaaa 271524_NNK_HsCas12a_K524 ccagaaactacgccaccaagNNKccctacagcgtggaaaagtt 272525_NNK_HsCas12a_P525 ggccagaaactacgccaccaagaaaNNKtacagcgtggaaaagtttaag273 526_NNK_HsCas12a_Y526 aactacgccaccaagaaacccNNKagcgtggaaaagtttaagctg274 527_NNK_HsCas12a_S527ctacgccaccaagaaaccctacNNKgtggaaaagtttaagctgaactt 275528_NNK_HsCas12a_V528 cgccaccaagaaaccctacagcNNKgaaaagtttaagctgaacttcc276 529_NNK_HsCas12a_E529caccaagaaaccctacagcgtgNNKaagtttaagctgaacttccaga 277530_NNK_HsCas12a_K530 caagaaaccctacagcgtggaaNNKtttaagctgaacttccagatgc278 531_NNK_HsCas12a_F531 aaaccctacagcgtggaaaagNNKaagctgaacttccagatgccc279 532_NNK_HsCas12a_K532 ccctacagcgtggaaaagtttNNKctgaacttccagatgcccac280 533_NNK_HsCas12a_L533 tacagcgtggaaaagtttaagNNKaacttccagatgcccaccctg281 534_NNK_HsCas12a_N534 gcgtggaaaagtttaagctgNNKttccagatgcccaccctggc282 535_NNK_HsCas12a_F535 cgtggaaaagtttaagctgaacNNKcagatgcccaccctggccag283 536_NNK_HsCas12a_Q536 ggaaaagtttaagctgaacttcNNKatgcccaccctggccagcg284 537_NNK_HsCas12a_M537 ttaagctgaacttccagNNKcccaccctggccagcgg 285538_NNK_HsCas12a_P538 aagctgaacttccagatgNNKaccctggccagcggct 286539_NNK_HsCas12a_T539 gaacttccagatgcccNNKctggccagcggctggg 287540_NNK_HsCas12a_L540 ttccagatgcccaccNNKgccagcggctgggac 288541_NNK_HsCas12a_A541 cagatgcccaccctgNNKagcggctgggacgtg 289542_NNK_HsCas12a_S542 atgcccaccctggccNNKggctgggacgtgaacaa 290543_NNK_HsCas12a_G543 gcccaccctggccagcNNKtgggacgtgaacaaag 291544_NNK_HsCas12a_W544 ccaccctggccagcggcNNKgacgtgaacaaagagaag 292545_NNK_HsCas12a_D545 ccctggccagcggctggNNKgtgaacaaagagaagaac 293546_NNK_HsCas12a_V546 ctggccagcggctgggacNNKaacaaagagaagaacaacg 294547_NNK_HsCas12a_N547 ccagcggctgggacgtgNNKaaagagaagaacaacggc 295548_NNK_HsCas12a_K548 gcggctgggacgtgaacNNKgagaagaacaacggcgc 296549_NNK_HsCas12a_E549 cggctgggacgtgaacaaaNNKaagaacaacggcgccatc 297550_NNK_HsCas12a_K550 ctgggacgtgaacaaagagNNKaacaacggcgccatcctgt 298551_NNK_HsCas12a_N551 gggacgtgaacaaagagaagNNKaacggcgccatcctgttcg 299552_NNK_HsCas12a_N552 gacgtgaacaaagagaagaacNNKggcgccatcctgttcgtgaa 300553_NNK_HsCas12a_G553 cgtgaacaaagagaagaacaacNNKgccatcctgttcgtgaagaacg301 554_NNK_HsCas12a_A554gaacaaagagaagaacaacggcNNKatcctgttcgtgaagaacggac 302555_NNK_HsCas12a_I555 agagaagaacaacggcgccNNKctgttcgtgaagaacggac 303556_NNK_HsCas12a_L556 gagaagaacaacggcgccatcNNKttcgtgaagaacggactgtac 304557_NNK_HsCas12a_F557 gaacaacggcgccatcctgNNKgtgaagaacggactgtact 305558_NNK_HsCas12a_V558 caacggcgccatcctgttcNNKaagaacggactgtactacc 306559_NNK_HsCas12a_K559 ggcgccatcctgttcgtgNNKaacggactgtactacctg 307560_NNK_HsCas12a_N560 gccatcctgttcgtgaagNNKggactgtactacctgggc 308561_NNK_HsCas12a_G561 gccatcctgttcgtgaagaacNNKctgtactacctgggcatcatg 309562_NNK_HsCas12a_L562 tcctgttcgtgaagaacggaNNKtactacctgggcatcatgcc 310563_NNK_HsCas12a_Y563 ctgttcgtgaagaacggactgNNKtacctgggcatcatgcctaag 311564_NNK_HsCas12a_Y564 cgtgaagaacggactgtacNNKctgggcatcatgcctaagc 312565_NNK_HsCas12a_L565 gtgaagaacggactgtactacNNKggcatcatgcctaagcagaag 313566_NNK_HsCas12a_G566 aagaacggactgtactacctgNNKatcatgcctaagcagaagggc 314567_NNK_HsCas12a_I567 ggactgtactacctgggcNNKatgcctaagcagaagggc 315568_NNK_HsCas12a_M568 gactgtactacctgggcatcNNKcctaagcagaagggcagata 316569_NNK_HsCas12a_P569 ctgtactacctgggcatcatgNNKaagcagaagggcagatacaag 317570_NNK_HsCas12a_K570 ctacctgggcatcatgcctNNKcagaagggcagatacaagg 318571_NNK_HsCas12a_Q571 cctgggcatcatgcctaagNNKaagggcagatacaaggccc 319572_NNK_HsCas12a_K572 ggcatcatgcctaagcagNNKggcagatacaaggccctg 320573_NNK_HsCas12a_G573 ggcatcatgcctaagcagaagNNKagatacaaggccctgtccttt 321574_NNK_HsCas12a_R574 catgcctaagcagaagggcNNKtacaaggccctgtcctttg 322575_NNK_HsCas12a_Y575 gcctaagcagaagggcagaNNKaaggccctgtcctttgagc 323576_NNK_HsCas12a_K576 aagcagaagggcagatacNNKgccctgtcctttgagccc 324577_NNK_HsCas12a_A577 gcagaagggcagatacaagNNKctgtcctttgagcccaccg 325578_NNK_HsCas12a_L578 gaagggcagatacaaggccNNKtcctttgagcccaccgaaa 326579_NNK_HsCas12a_S579 gggcagatacaaggccctgNNKtttgagcccaccgaaaaga 327580_NNK_HsCas12a_F580 agatacaaggccctgtccNNKgagcccaccgaaaagacc 328581_NNK_HsCas12a_E581 ggcagatacaaggccctgtcctttNNKcccaccgaaaagac 329582_NNK_HsCas12a_P582 caaggccctgtcctttgagNNKaccgaaaagaccagcgag 330584_NNK_HsCas12a_E584 ctgtcctttgagcccaccNNKaagaccagcgagggcttt 331584_NNK_HsCas12a_T584 ccctgtcctttgagcccNNKgaaaagaccagcgaggg 332585_NNK_HsCas12a_K585 gtcctttgagcccaccgaaNNKaccagcgagggctttg 333586_NNK_HsCas12a_T586 tgtcctttgagcccaccgaaaagNNKagcgagggctttgac 334587_NNK_HsCas12a_S587 tttgagcccaccgaaaagaccNNKgagggctttgacaagatgtac 335588_NNK_HsCas12a_E588 gagcccaccgaaaagaccagcNNKggctttgacaagatgtactac 336589_NNK_HsCas12a_G589 cccaccgaaaagaccagcgagNNKtttgacaagatgtactacgat 337590_NNK_HsCas12a_F590 accgaaaagaccagcgagggcNNKgacaagatgtactacgattact 338591_NNK_HsCas12a_D591 ccgaaaagaccagcgagggctttNNKaagatgtactacgattacttcc339 592_NNK_HsCas12a_K592 aagaccagcgagggctttgacNNKatgtactacgattacttcccc340 593_NNK_HsCas12a_M593 ccagcgagggctttgacaagNNKtactacgattacttccccga341 594_NNK_HsCas12a_Y594 gcgagggctttgacaagatgNNKtacgattacttccccgacgc342 595_NNK_HsCas12a_Y595 gggctttgacaagatgtacNNKgattacttccccgacgccg 343596_NNK_HsCas12a_D596 ggctttgacaagatgtactacNNKtacttccccgacgccgccaa 344597_NNK_HsCas12a_Y597 agggctttgacaagatgtactacgatNNKttccccgacgccgc 345598_NNK_HsCas12a_F598 gctttgacaagatgtactacgattacNNKcccgacgccgccaaga 346599_NNK_HsCas12a_P599 tttgacaagatgtactacgattacttcNNKgacgccgccaagatgatc347 600_NNK_HsCas12a_D600 gtactacgattacttccccNNKgccgccaagatgatcccca 348601_NNK_HsCas12a_A601 actacgattacttccccgacNNKgccaagatgatccccaagtg 349602_NNK_HsCas12a_A602 cgattacttccccgacgccNNKaagatgatccccaagtgca 350603_NNK_HsCas12a_K603 cttccccgacgccgccNNKatgatccccaagtgca 351604_NNK_HsCas12a_M604 cccgacgccgccaagNNKatccccaagtgcagc 352605_NNK_HsCas12a_I605 cgacgccgccaagatgNNKcccaagtgcagcaccc 353606_NNK_HsCas12a_P606 gacgccgccaagatgatcNNKaagtgcagcacccagctg 354607_NNK_HsCas12a_K607 ccgccaagatgatccccNNKtgcagcacccagctgaa 355608_NNK_HsCas12a_C608 gccaagatgatccccaagNNKagcacccagctgaaggcc 356609_NNK_HsCas12a_S609 aagatgatccccaagtgcNNKacccagctgaaggccgtg 357610_NNK_HsCas12a_T610 tgatccccaagtgcagcNNKcagctgaaggccgtgac 358611_NNK_HsCas12a_Q611 ccccaagtgcagcaccNNKctgaaggccgtgaccg 359613_NNK_HsCas12a_K613 tgcagcacccagctgNNKgccgtgaccgcccac 360613_NNK_HsCas12a_L613 caagtgcagcacccagNNKaaggccgtgaccgccc 361614_NNK_HsCas12a_A614 gcagcacccagctgaagNNKgtgaccgcccactttca 362615_NNK_HsCas12a_V615 gcacccagctgaaggccNNKaccgcccactttcagac 363616_NNK_HsCas12a_T616 ccagctgaaggccgtgNNKgcccactttcagaccc 364617_NNK_HsCas12a_A617 cagctgaaggccgtgaccNNKcactttcagacccacacc 365618_NNK_HsCas12a_H618 tgaaggccgtgaccgccNNKtttcagacccacaccac 366619_NNK_HsCas12a_F619 gccgtgaccgcccacNNKcagacccacaccacc 367620_NNK_HsCas12a_Q620 aaggccgtgaccgcccactttNNKacccacaccacc 368621_NNK_HsCas12a_T621 gaccgcccactttcagNNKcacaccacccccatcc 369622_NNK_HsCas12a_H622 cgcccactttcagaccNNKaccacccccatcctgc 370623_NNK_HsCas12a_T623 cccactttcagacccacNNKacccccatcctgctgag 371624_NNK_HsCas12a_T624 cactttcagacccacaccNNKcccatcctgctgagcaac 372625_NNK_HsCas12a_P625 cactttcagacccacaccaccNNKatcctgctgagcaacaacttc 373626_NNK_HsCas12a_I626 cagacccacaccacccccNNKctgctgagcaacaacttc 374627_NNK_HsCas12a_L627 gacccacaccacccccatcNNKctgagcaacaacttcatcg 375628_NNK_HsCas12a_L628 ccacaccacccccatcctgNNKagcaacaacttcatcgagc 376629_NNK_HsCas12a_S629 accacccccatcctgctgNNKaacaacttcatcgagccc 377630_NNK_HsCas12a_N630 cccccatcctgctgagcNNKaacttcatcgagcccct 378631_NNK_HsCas12a_N631 cccatcctgctgagcaacNNKttcatcgagcccctggaa 379632_NNK_HsCas12a_F632 ccatcctgctgagcaacaacNNKatcgagcccctggaaatcac 380633_NNK_HsCas12a_I633 cctgctgagcaacaacttcNNKgagcccctggaaatcacca 381634_NNK_HsCas12a_E634 cctgctgagcaacaacttcatcNNKcccctggaaatcaccaaagag 382635_NNK_HsCas12a_P635 gctgagcaacaacttcatcgagNNKctggaaatcaccaaagagatct383 636_NNK_HsCas12a_L636gagcaacaacttcatcgagcccNNKgaaatcaccaaagagatctacg 384637_NNK_HsCas12a_E637 aacaacttcatcgagcccctgNNKatcaccaaagagatctacgac 385638_NNK_HsCas12a_I638 aacaacttcatcgagcccctggaaNNKaccaaagagatctacgacc 386639_NNK_HsCas12a_T639 ttcatcgagcccctggaaatcNNKaaagagatctacgacctgaac 387640_NNK_HsCas12a_K640 cgagcccctggaaatcaccNNKgagatctacgacctgaaca 388840_NNK_HsCas12a_D840 ctgagccacgacctgtccNNKgaagctagagcactgctg 389841_NNK_HsCas12a_E841 ccacgacctgtccgacNNKgctagagcactgctgc 390842_NNK_HsCas12a_A842 ccacgacctgtccgacgaaNNKagagcactgctgccc 391843_NNK_HsCas12a_R843 gacctgtccgacgaagctNNKgcactgctgcccaacg 392844_NNK_HsCas12a_A844 ctgtccgacgaagctagaNNKctgctgcccaacgtgatc 393845_NNK_HsCas12a_L845 gtccgacgaagctagagcaNNKctgcccaacgtgatcacaa 394846_NNK_HsCas12a_L846 ccgacgaagctagagcactgNNKcccaacgtgatcacaaaaga 395847_NNK_HsCas12a_P847 gacgaagctagagcactgctgNNKaacgtgatcacaaaagaggtg 396848_NNK_HsCas12a_N848 aagctagagcactgctgcccNNKgtgatcacaaaagaggtgtc 397849_NNK_HsCas12a_V849 ctagagcactgctgcccaacNNKatcacaaaagaggtgtccca 398850_NNK_HsCas12a_I850 gcactgctgcccaacgtgNNKacaaaagaggtgtcccac 399851_NNK_HsCas12a_T851 ctgctgcccaacgtgatcNNKaaagaggtgtcccacgag 400852_NNK_HsCas12a_K852 ctgcccaacgtgatcacaNNKgaggtgtcccacgagatc 401853_NNK_HsCas12a_E853 cactgctgcccaacgtgatcacaaaaNNKgtgtcccacgagat 402854_NNK_HsCas12a_V854 gcccaacgtgatcacaaaagagNNKtcccacgagatcatcaaggac 403855_NNK_HsCas12a_S855 aacgtgatcacaaaagaggtgNNKcacgagatcatcaaggaccgg 404856_NNK_HsCas12a_H856 tgatcacaaaagaggtgtccNNKgagatcatcaaggaccggcg 405857_NNK_HsCas12a_E857 cacaaaagaggtgtcccacNNKatcatcaaggaccggcggt 406858_NNK_HsCas12a_I858 caaaagaggtgtcccacgagNNKatcaaggaccggcggtttac 407859_NNK_HsCas12a_I859 gaggtgtcccacgagatcNNKaaggaccggcggtttacc 408860_NNK_HsCas12a_K860 gtgtcccacgagatcatcNNKgaccggcggtttacctc 409861_NNK_HsCas12a_D861 gtgtcccacgagatcatcaagNNKcggcggtttacctcc 410862_NNK_HsCas12a_R862 tcccacgagatcatcaaggacNNKcggtttacctccgataagttc 411863_NNK_HsCas12a_R863 cacgagatcatcaaggaccggNNKtttacctccgataagttcttc 412864_NNK_HsCas12a_F864 gagatcatcaaggaccggcggNNKacctccgataagttcttcttc 413865_NNK_HsCas12a_T865 cgagatcatcaaggaccggcggtttNNKtccgataagttcttcttc 414866_NNK_HsCas12a_S866 atcaaggaccggcggtttaccNNKgataagttcttcttccacgtg 415867_NNK_HsCas12a_D867 ggaccggcggtttacctccNNKaagttcttcttccacgtgc 416868_NNK_HsCas12a_K868 ccggcggtttacctccgatNNKttcttcttccacgtgccc 417869_NNK_HsCas12a_F869 ggcggtttacctccgataagNNKttcttccacgtgcccatcac 418870_NNK_HsCas12a_F870 ggtttacctccgataagttcNNKttccacgtgcccatcaccct 419871_NNK_HsCas12a_F871 tttacctccgataagttcttcNNKcacgtgcccatcaccctgaac 420872_NNK_HsCas12a_H872 acctccgataagttcttcttcNNKgtgcccatcaccctgaactac 421873_NNK_HsCas12a_V873 tccgataagttcttcttccacNNKcccatcaccctgaactaccag 422874_NNK_HsCas12a_P874 gataagttcttcttccacgtgNNKatcaccctgaactaccaggcc 423875_NNK_HsCas12a_I875 ttcttcttccacgtgcccNNKaccctgaactaccaggcc 424876_NNK_HsCas12a_T876 tcttccacgtgcccatcNNKctgaactaccaggccgc 425877_NNK_HsCas12a_L877 ccacgtgcccatcaccNNKaactaccaggccgcca 426878_NNK_HsCas12a_N878 cgtgcccatcaccctgNNKtaccaggccgccaaca 427879_NNK_HsCas12a_Y879 gcccatcaccctgaacNNKcaggccgccaacagcc 428880_NNK_HsCas12a_Q880 ccatcaccctgaactacNNKgccgccaacagcccca 429881_NNK_HsCas12a_A881 atcaccctgaactaccagNNKgccaacagccccagcaag 430882_NNK_HsCas12a_A882 accctgaactaccaggccNNKaacagccccagcaagttc 431883_NNK_HsCas12a_N883 ctgaactaccaggccgccNNKagccccagcaagttcaac 432884_NNK_HsCas12a_S884 aactaccaggccgccaacNNKcccagcaagttcaaccag 433885_NNK_HsCas12a_P885 ctaccaggccgccaacagcNNKagcaagttcaaccagagag 434886_NNK_HsCas12a_S886 caggccgccaacagccccNNKaagttcaaccagagagtg 435887_NNK_HsCas12a_K887 gccgccaacagccccagcNNKttcaaccagagagtgaac 436888_NNK_HsCas12a_F888 gccaacagccccagcaagNNKaaccagagagtgaacgcc 437889_NNK_HsCas12a_N889 caacagccccagcaagttcNNKcagagagtgaacgcctacc 438890_NNK_HsCas12a_Q890 cagccccagcaagttcaacNNKagagtgaacgcctacctga 439891_NNK_HsCas12a_R891 ccccagcaagttcaaccagNNKgtgaacgcctacctgaaag 440892_NNK_HsCas12a_V892 cccagcaagttcaaccagagaNNKaacgcctacctgaaagagcac 441893_NNK_HsCas12a_N893 gcaagttcaaccagagagtgNNKgcctacctgaaagagcaccc 442894_NNK_HsCas12a_A894 aagttcaaccagagagtgaacNNKtacctgaaagagcaccccgag 443895_NNK_HsCas12a_Y895 aaccagagagtgaacgccNNKctgaaagagcaccccgag 444896_NNK_HsCas12a_L896 ccagagagtgaacgcctacNNKaaagagcaccccgagacac 445897_NNK_HsCas12a_K897 gagtgaacgcctacctgNNKgagcaccccgagacacc 446898_NNK_HsCas12a_E898 agagagtgaacgcctacctgaaaNNKcaccccgagacacc 447899_NNK_HsCas12a_H899 gaacgcctacctgaaagagNNKcccgagacacccatcattg 448900_NNK_HsCas12a_P900 cgcctacctgaaagagcacNNKgagacacccatcattggca 449901_NNK_HsCas12a_E901 ctacctgaaagagcaccccNNKacacccatcattggcatcg 450902_NNK_HsCas12a_T902 ctgaaagagcaccccgagNNKcccatcattggcatcgac 451903_NNK_HsCas12a_P903 tgaaagagcaccccgagacaNNKatcattggcatcgacagagg 452904_NNK_HsCas12a_I904 agcaccccgagacacccNNKattggcatcgacagagg 453905_NNK_HsCas12a_I905 ccccgagacacccatcNNKggcatcgacagaggcg 454906_NNK_HsCas12a_G906 gagcaccccgagacacccatcattNNKatcgacagaggcg 455907_NNK_HsCas12a_I907 gagacacccatcattggcNNKgacagaggcgagcggaac 456908_NNK_HsCas12a_D908 acacccatcattggcatcNNKagaggcgagcggaacctg 457909_NNK_HsCas12a_R909 cccatcattggcatcgacNNKggcgagcggaacctgatc 458910_NNK_HsCas12a_G910 cccatcattggcatcgacagaNNKgagcggaacctgatctacatc 459911_NNK_HsCas12a_E911 tcattggcatcgacagaggcNNKcggaacctgatctacatcac 460912_NNK_HsCas12a_R912 tggcatcgacagaggcgagNNKaacctgatctacatcaccg 461913_NNK_HsCas12a_N913 atcgacagaggcgagcggNNKctgatctacatcaccgtg ^(a)For“NNK”, N refers to A, C, T or G; K refers to G or T.

With respect to Table 4, the reference (i.e., wild-type) polypeptidesequence is SEQ ID NO.: 462 upon which these mutants are based bycomparison. Polynucleotides codon-optimized for expression in E. coliand human cells that encode SEQ ID NO.:462 are SEQ ID NOs.: 463 and 464,respectively. The same mutations were introduced as well intoM537R/F870L-AsCas12a background. The corresponding reference polypeptidesequence for the M537R/F870L-Cas12a is SEQ ID NO.: 465 (the alteredamino acids are underlined). Polynucleotides codon-optimized forexpression in E. coli and human cells that encode SEQ ID NO.:465 are SEQID NOs.: 466 and 467, respectively (the altered codons are underlined).

SEQ ID NO.: 462 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRN SEQ ID NO.:463 atgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCA TATATCCAAGAACTGCGTAACSEQ ID NO.: 464 atgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCA TATATCCAAGAACTGCGTAACSEQ ID NO.: 465 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRN SEQ ID NO.:466 atgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCCTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCA TATATCCAAGAACTGCGTAACSEQ ID NO.: 467 atgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCCTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCA TATATCCAAGAACTGCGTAAC

Additional polynucleotides and polypeptides relevant to this Exampleinclude Cas12a variants having single amino acid substitution at M537Rand F870L, as shown below. The underlined codons or amino acidscorrespond to the changes relative to the corresponding WT Cas12asequences.

SEQ ID NO.: 468 E. coli optimized DNA M537RatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACSEQ ID NO.: 469 E. coli optimized DNA F870L Cas12aatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCCTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACSEQ ID NO.: 470 Human optimized DNA M537R Cas12aatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACSEQ ID NO.: 471 Human optimized DNA F870L Cas12aatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCCTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACSEQ ID NO.: 472 M537R Cas12a AAMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNSEQ ID NO.: 473 F870L Cas12a AAMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN

Example 6 Rational Design of Fusion Cas12a Polypeptides

Fusion Cas12/a polypeptides having additional motifs enabling nuclearlocalization into eukaryotic cells (collectively, “NLS” or “NLSsequences”), and/or purification and label detection motifs(collectively, “affinity tags”) fall within the scope of the presentinvention. Exemplary nuclear localization signals (“NLS” or “NLSsequences”) are well known in the art and include those listedidentified by polynucleotide and amino acid sequences depicted in Table6.

TABLE 6 Exemplary NLS sequences SEQ ID NLS NO.: NameSequence (5′→3′ or N→C) 474 SV40 CCGAAAAAAAAACGTAAAGTTGG 475 SV40PKKKRKV 476 OpT AGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTT 477 OpT SSDDEATADSQHAAPPKKKRKV 478 aNLSCCGCCTCCGAAACGTCCGCGTCTGGAT 479 aNLS PPPKRPRLD 480 BIP1AAACGTCCGGCAGCAACCAAAAAAGCAGGTCAGGC AAAAAAGAAAAAA 481 BIP1KRPAATKKAGQAKKKK 482 BIP2 AAACGTACCGCAGATGGTAGCGAATTTGAAAGCCCGAAAAAAAAGCGTAAGGTGGAA 483 BIP2 KRTADGSEFESPKKKRKVE

Exemplary purification and/or label detection motifs include affinitytags that are also well known in the art. Often, additional amino acidlinkers inserted before or after the additional motifs can provideimprovements in expression and/or stability of the expressed fusionCas12/a polypeptide. Two examples of affinity tags are defined bypolynucleotide and amino acid sequences depicted below in Table 7.

TABLE 7 Exemplary affinity tags SEQ ID Affinity Tag NO.: NameSequence (5′→3′ or N→C) 484 V5 GGTAAACCGATTCCGAATCCGCTGCTGGGTCTGGATAGCACC 485 V5 GKPIPNPLLGLDST 486 HIS CACCACCACCACCACCAC 487 HISHHHHHH

Fusion Cas12a polypeptides that include a nuclear localization signal,linker amino acids and/or affinity tags can be readily constructed usingchemical polypeptide methods or expressed from engineeredpolynucleotides encoding in-frame polypeptides created with recombinantDNA technology. Such technologies are well known and within the purviewof one skilled in the art. Working examples of such polynucleotides andpolypeptides are illustrated by SEQ ID NOs.: 5-30. Fusion Cas12apolypeptide variants that encode the open reading frames of SEQ ID NOs.:59-245 having nuclear localization sequences and/or affinity tags andoptionally amino acid linkers as needed fall within the scope of thisdisclosure. Exemplary Cas12a variants having nuclear localizationsignals are presented below.

Briefly, the method of site directed mutagenesis (SDM) was employed tocreate the expression constructs having As Cas12a coding sequences withdifferent nuclear localization signals (NLS's). Site directedmutagenesis was performed by designing complimentary primers thatencompass the desired nucleotide base change(s), along with flankingplasmid vector sequence, wherein each flanking region has a meltingtemperature (T_(m)) of at least 60° C. A polymerase chain reaction (PCR)run was then performed using standard cycling conditions for a total of16 cycles. The restriction enzyme, DPN I, was added to digest away thestarting plasmid vector material so only the new product containing thebase changes remain. After DPN I treatment, a small amount of the PCRproduct was transformed into competent E. coli cells, recovered in SOCmedia and plated onto kanamycin resistance Luria Broth (LB) agar plates.Colonies were screened using the Sanger sequencing method to verifycorrect base changes in selected clones.

SEQ ID NO.: 488 E. coli optimized DNA WT Cas12a with NLS linkersatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGT AGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTT GGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGT GGCTCTGGTGGTAGCctcgagcaccaccaccaccaccac

The underlined sequences refer to nucleotides encoding amino acid linkersequences. The double-underlined sequences refer to nucleotides encodingnuclear localization sequences (NLS linker). The italicized sequencesrefer to nucleotides encoding amino acid affinity tag sequences((HIS)₆).

SEQ ID NO. 489 E. coli optimized DNA M537R F870L Cas12a with NLS linkersatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGA ACTTTCAG CGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGA CAAATTC CTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGT AGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTT GGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGT GGCTCTGGTGGTAGCctcgagcaccaccaccaccaccac

The bolded and underlined sequences refer to mutant codons introducedinto the Cas12a open reading frame. The underlined sequences refer tonucleotides encoding amino acid linker sequences. The double-underlinedsequences refer to nucleotides encoding nuclear localization sequences(NLS linker). The italicized sequences refer to nucleotides encodingamino acid affinity tag sequences ((HIS)₆).

SEQ ID NO.: 490 Human optimized DNA WT Cas12a with NLS linkersatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGT AGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTT GGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGT GGCTCTGGTGGTAGCctcgagcaccaccaccaccaccac

The underlined sequences refer to nucleotides encoding amino acid linkersequences. The double-underlined sequences refer to nucleotides encodingnuclear localization sequences (NLS linker). The italicized sequencesrefer to nucleotides encoding amino acid affinity tag sequences((HIS)₆).

SEQ ID NO.: 491 Human optimized DNA M537R F870L Cas12a with NLS linkersatgACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGA ACTTTCAG CGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGA CAAATTC CTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACGGTCGT AGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGCACCGCCTAAAAAGAAACGTAAAGTT GGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTGGTGGCTCAGGGGGTTCTGGT GGCTCTGGTGGTAGCctcgagcaccaccaccaccaccac

The bolded and underlined sequences refer to mutant codons introducedinto the Cas12a open reading frame. The underlined sequences refer tonucleotides encoding amino acid linker sequences. The double-underlinedsequences refer to nucleotides encoding nuclear localization sequences(NLS linker). The italicized sequences refer to nucleotides encodingamino acid affinity tag sequences ((HIS)₆).

SEQ ID NO.: 492 WT Cas12a AA with NLS linkersMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRNGRSSDDEATADSQHAAPPKKKRKV GGSGGSGGSGGSGGSGGSG GSGGSLE HHHHHH

The underlined sequences refer to amino acid sequences encoding aminoacid linker sequences. The double-underlined sequences refer to aminoacid sequences encoding nuclear localization sequences (NLS linker). Theitalicized sequences refer to amino acid sequences encoding amino acidaffinity tag sequences ((HIS)₆).

SEQ ID NO.: 493 M537R F870L Cas12a AA with NLS linkersMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQ R PTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVI TKEVSHEIIKDRRFTSDKF LFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRNGRSSDDEATADSQHAAPPKKKRKV GGSGGSGGSGGSGGSGGSG GSGGSLE HHHHHH

The bolded and underlined sequences refer to mutant amino acidsintroduced into the Cas12a polypeptide variant. The underlined sequencesrefer to amino acid sequences encoding amino acid linker sequences. Thedouble-underlined sequences refer to amino acid sequences encodingnuclear localization sequences (NLS linker). The italicized sequencesrefer to amino acid sequences encoding amino acid affinity tag sequences((HIS)₆).

REFERENCES

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Gao, L., et al., Engineered Cpf1 variants with altered PAM specificitiesincrease genome targeting range. Nat Biotechnol. 2017; 35(8): 789-792.

Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease inadaptive bacterial immunity. Science. 2012; 337:816-821. doi:10.1126/science.1225829.

Kleinstiver, B. P., et al., High-fidelity CRISPR-Cas9 nucleases with nodetectable genome-wide off-target effects. Nature, 2016. 529(7587): p.490-5.

Slaymaker, I. M., et al., Rationally engineered Cas9 nucleases withimproved specificity. Science, 2016. 351(6268): p. 84-8.

Sun, Y., et al., Factors influencing the nuclear targeting ability ofnuclear localization signals. J Drug Target, 2016. 24(10): p. 927-933.

Wrenbeck E E, Klesmith J R, Stapleton J A, Adeniran A, Tyo K E,Whitehead T A. Plasmid-based one-pot saturation mutagenesis. NatMethods. 2016; 13(11):928-930. doi:10.1038/nmeth.4029

Zetsche, B., et al., Cpf1 Is a Single RNA-Guided Endonuclease of a Class2 CRISPR-Cas System. Cell. 2015; 163:759-771. doi:10.1016/j.cell.2015.09.038.

INCORPORATION BY REFERENCE

All of the patents, patent applications, patent applicationpublications, and other publications cited herein are herebyincorporated by reference as if set forth in their entirety.

Preferred Embodiments

The present invention has been described in connection with what arepresently considered to be the most practical and preferred embodiments.However, the invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,one of skill in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

What is claimed is:
 1. A CRISPR-associated protein comprising a variantof a Cas12a, wherein the variant of the Cas12a comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 472, 473 and465.
 2. The CRISPR-associated protein according to claim 1, wherein thevariant of the Cas12a consists of SEQ ID NO:
 472. 3. TheCRISPR-associated protein according to claim 1, wherein the variant ofthe Cas12a consists of SEQ ID NO:
 473. 4. The CRISPR-associated proteinaccording to claim 1, wherein the variant of the Cas12a consists of SEQID NO:
 465. 5. A CRISPR ribonucleoprotein complex, comprising: a guideRNA; and a CRISPR-associated protein comprising a variant of a Cas12a,wherein the variant of the Cas12a comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 472, 473 and
 465. 6.The CRISPR ribonucleoprotein complex of claim 5, wherein the variant ofthe Cas12a consists of SEQ ID NO:
 472. 7. The CRISPR ribonucleoproteincomplex of claim 5, wherein the variant of the Cas12a consists of SEQ IDNO:
 473. 8. The CRISPR ribonucleoprotein complex of claim 5, wherein thevariant of the Cas12a consists of SEQ ID NO:
 465. 9. A CRISPR-associatedprotein comprising a variant of the Cas12a—of SEQ ID NO.: 462, whereinthe variant of the Cas12a comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO: 59-245.
 10. A Cas12a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:18-22 and 24-30.
 11. A CRISPR-associated protein comprising afusion polypeptide comprising a Cas12a protein, a nuclear localizationsignal, optionally an amino acid linker and optionally an affinity tag,wherein the Cas12a protein comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 59-245, and wherein the nuclearlocalization signal comprises an amino acid sequence selected from SEQID NO: 475, 477, 479, 481 and
 483. 12. A CRISPR-associated protein,wherein the CRISPR-associated protein is encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 489 and 491.13. A CRISPR-associated protein, wherein the CRISPR-associated proteincomprises SEQ ID NO: 493.